Nanopatterned Medical Device with Enhanced Cellular Interaction

ABSTRACT

Disclosed are nanotopography-based methods and devices for interacting with a component of the dermal connective tissue. Devices include structures fabricated on a surface to form a nanotopography. A random or non-random pattern of structures may be fabricated such as a complex pattern including structures of differing sizes and/or shapes. Microneedles may be beneficially utilized for delivery of an agent to a cell or tissue. Devices may be utilized to directly or indirectly alter cell behavior through the interaction of a fabricated nanotopography with the plasma membrane of a cell and/or with an extracellular matrix component.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/328,723 having a filing date of Apr. 28, 2010, U.S.Provisional Patent Application Ser. No. 61/411,071 having a filing dateof Nov. 8, 2010, and U.S. Provisional Patent Application Ser. No.61/435,939 having a filing date of Jan. 25, 2011, all of which areincorporated herein in their entirety by reference.

BACKGROUND

Targeted drug delivery in which an agent (e.g., a drug or a therapeutic)is provided in an active state to a specific cell or tissue type ateffective concentrations is a long sought goal. Many difficulties mustbe overcome to reach this goal. For instance, an agent must first besuccessfully delivered to the desired target. Primary delivery methodspresently used include oral delivery and injections. However, injectionsare painful and both methods tend to provide bursts of agents ratherthan a preferred steady-state delivery. Additionally, the human body hasdeveloped many systems to prevent the influx of foreign substances suchas enzymatic degradation in the gastrointestinal tract, structuralcomponents that prevent absorption across epithelium, hepatic clearance,and immune and foreign body response.

Transdermal delivery materials have been developed in an attempt toprovide a painless route for delivery of active agents over a sustainedperiod. In order to be successful, a transdermal scheme must deliver anagent across the epidermis, which has evolved with a primary function ofkeeping foreign substances out. The outermost layer of the epidermis,the stratum corneum, has structural stability provided by overlappingcorneocytes and crosslinked keratin fibers held together bycoreodesmosomes and embedded within a lipid matrix, all of whichprovides an excellent barrier function. Beneath the stratum corneum isthe stratum granulosum, within which tight junctions are formed betweenkeratinocytes. Tight junctions are barrier structures that include anetwork of transmembrane proteins embedded in adjacent plasma membranes(e.g., claudins, occludin, and junctional adhesion molecules) as well asmultiple plaque proteins (e.g., ZO-1, ZO-2, ZO-3, cingulin, symplekin).Tight junctions are found in internal epithelium (e.g., the intestinalepithelium, the blood-brain barrier) as well as in the stratumgranulosum of the skin. Beneath both the stratum corneum and the stratumgranulosum lays the stratum spinosum. The stratum spinosum includesLangerhans cells, which are dendritic cells that may become fullyfunctioning antigen-presenting cells and may institute an immuneresponse and/or a foreign body response to an invading agent.

In spite of the difficulties of crossing the natural boundaries,progress has been made in attaining delivery of active agents, e.g.,transdermal delivery. Unfortunately, transdermal delivery methods arepresently limited to delivery of low molecular weight agents that have amoderate lipophilicity and no charge. Even upon successful crossing ofthe natural boundary, problems still exist with regard to maintainingthe activity level of delivered agents and avoidance of foreign body andimmune response.

The utilization of supplementary methods to facilitate transdermaldelivery of active agents has improved this delivery route. Forinstance, microneedle devices have been found to be useful in transportof material into or across the skin. In general, a microneedle deviceincludes an array of needles that may penetrate the stratum corneum ofthe skin and reach an underlying layer. Examples of microneedle deviceshave been described in U.S. Pat. No. 6,334,856 to Allen, et at and U.S.Pat. No. 7,226,439 to Prausnitz, et al., both of which are incorporatedherein by reference. However, as discussed above, transdermal deliverypresents additional difficulties beyond the barrier of the stratumcorneum. In particular, once an agent has been delivered to a targetedarea, it is still necessary that proper utilization take place withoutdestruction of the agent or the instigation of an immune response. Forinstance, encouraging endocytosis of an active agent targeted to thecell interior presents difficulties.

Researchers have gained understanding of the molecular world in whichdelivery activities occur in an attempt to overcome such problems. Forinstance, chitosan has been found to be effective in opening tightjunctions in the intestinal epithelium (see, e.g., Sapra, et al., AAPSPharm. Sci. Tech., 10(1), March, 2009; Kaushal, et al., Sci. Pharm.,2009; 77; 877-897), and delivery of active agents via endocytosis oflabeled nanoparticles has been described (see, e.g., U.S. Pat. Nos.7,563,451 to Lin, et al. and 7,544,770 to Haynie). In addition, thenanotopography of a surface adjacent to a cell has been found to affectadhesive characteristics between the two as well as to effect cellbehavior including morphology, motility, cytoskeleton architecture,proliferation, and differentiation (see, e.g., Hart, et al., EuropeanCells and Materials, Vol. 10, Suppl. 2, 2005; Lim et al., J R SocInterface, Mar. 22, 2005, 2(2), 97-108; Yim, et al., Biomaterials,September, 2005, 26(26), 5405-5413). As an extension of this initialresearch, nanotopography of supporting substrates has been examined foruse in tissue engineering (see, e.g., U.S. Patent ApplicationPublication Nos. 2008/0026464 to Borenstein, et al. and 2008/0311172 toSchapira, et al.).

While the above describe improvements in the art, further room forimprovement exists. For instance, devices and methods that provideefficient delivery of active agents while decreasing potential immuneand foreign body response to both the delivery device and the deliveredagents would be beneficial.

SUMMARY

In accordance with one embodiment of the present invention, a medicaldevice is disclosed that comprises an array of microneedles that extendoutwardly from a support. At least one of the microneedles contains aplurality of nanostructures formed on a surface thereof, thenanostructures being arranged in a predetermined pattern.

In accordance with another embodiment of the present invention, a methodfor delivering a drug compound to a subdermal location is disclosed. Themethod comprises penetrating the stratum corneum with a microneedle thatis in fluid communication with the drug compound, the microneedlecontaining a plurality of nanostructures formed on a surface thereof andarranged in a pattern; and transporting the drug compound through themicroneedle and across the stratum corneum.

In yet another embodiment of the present invention, a medical device isdisclosed that comprises a plurality of nano-sized structures that havebeen fabricated on the surface and define a fabricated nanotopography.Also disclosed is a method for forming a medical device that comprisesfabricating a pattern of nanostructures on a surface of a microneedle.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the subject matter, including the bestmode thereof, directed to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification, which makesreference to the appended figures in which:

FIG. 1 illustrates one embodiment of a microneedle device.

FIG. 2 illustrates another embodiment of a microneedle device.

FIG. 3 illustrates one embodiment of a microneedle including a surfacethat defines a nanotopography that may interact with an extracellularmatrix (ECM).

FIG. 4 illustrates one embodiment of a complex pattern that may beformed on a microneedle surface.

FIG. 5 illustrates a pattern including multiple iterations of thecomplex pattern of FIG. 4.

FIG. 6 illustrates a Sierpinski triangle fractal.

FIGS. 7A-7D illustrate complex fractal and fractal-likenanotopographies.

FIG. 8 illustrates another complex pattern that may be formed on amicroneedle surface.

FIG. 9 illustrates exemplary packing densities as may be utilized fornano-sized structures as described herein including a square packingdesign (FIG. 9A), a hexagonal packing design (FIG. 9B), and a circlepacking design (FIG. 9C).

FIG. 10 illustrates a method for determining the TEER of a cellularlayer.

FIGS. 11A-11C schematically illustrate a nanoimprinting method as may beutilized in one embodiment in forming a device.

FIG. 12 schematically illustrates one embodiment of a device.

FIG. 13 is a perspective view of one embodiment of a transdermal patchprior to delivery of a drug compound.

FIG. 14 is a front view of the patch of FIG. 13.

FIG. 15 is a perspective view of the patch of FIG. 13 in which therelease member is partially withdrawn from the patch.

FIG. 16 is a front view of the patch of FIG. 13.

FIG. 17 is a perspective view of the transdermal patch of FIG. 13 afterremoval of the release member and during use.

FIG. 18 is a front view of the patch of FIG. 17.

FIG. 19 is a perspective view of another embodiment of a transdermalpatch prior to delivery of a drug compound.

FIG. 20 is a front view of the patch of FIG. 19.

FIG. 21 is a perspective view of the patch of FIG. 19 in which therelease member is partially peeled away from the patch.

FIG. 22 is a front view of the patch of FIG. 21.

FIG. 23 is a perspective view of the patch of FIG. 19 in which therelease member is completely peeled away from the patch.

FIG. 24 is a perspective view of the transdermal patch of FIG. 19 afterremoval of the release member and during use.

FIGS. 25A-25E illustrate several nanotopography patterns as describedherein.

FIG. 26 is an SEM of a film including a nanopatterned surface.

FIGS. 27A and 27B are two SEM of a film including another nanopatternedsurface.

FIG. 28 is an SEM of a film including another nanopatterned surface.

FIG. 29 is an SEM of a film including another nanopatterned surface.

FIG. 30 is an SEM of a film including another nanopatterned surface.

FIG. 31 is an SEM of a film including another nanopatterned surface.

FIG. 32 is an SEM of a film including another nanopatterned surface.

FIG. 33 is an SEM of a film including another nanopatterned surface.

FIG. 34 is an SEM of a film including another nanopatterned surface.

FIG. 35 graphically illustrates the effects on permeability to bovineserum albumin (BSA) in a monolayer of cells on polystyrene filmspatterned with nanopatterns as described herein.

FIGS. 36A and 36B graphically illustrate the effects on permeability toimmunoglobulin-G (IgG) in a monolayer of cells on polystyrene filmspatterned with nanopatterns as described herein.

FIGS. 37A and 37B are 3D live/dead flourescein staining images showingparacellular and transcellular transport of IgG across a monolayer ofcells on a polystyrene patterned surface as described herein.

FIG. 38 graphically illustrates the effects on permeability to BSA in amonolayer of cells on polypropylene films patterned with nanopatterns asdescribed herein.

FIG. 39 graphically illustrates the effects on permeability to IgG in amonolayer of cells on polypropylene films patterned with nanopatterns asdescribed herein.

FIGS. 40A and 40B are 3D live/dead flourescein staining images showingparacellular transport of IgG across a monolayer of cells on apolypropylene patterned surface as described herein.

FIGS. 41A-41F are scanning electron microscopy (SEM) images of cellscultured on nanopatterned surfaces as described herein.

FIG. 42 illustrates the effects on permeability to etanercept in amonolayer of cells on polypropylene or polystyrene films patterns withnanopatterns as described herein.

FIG. 43 illustrates the increase in permeability to etanercept of acellular layer following two hours of contact with a polypropylene orpolystyrene films patterns with nanopatterns as described herein.

FIG. 44 is an array of microneedles including a surface layer defining apattern of nanostructures thereon.

FIG. 45 is a single microneedle of the array of FIG. 44.

FIG. 46 graphically illustrates the PK profile of a protein therapeuticdelivered with a device as described herein.

FIGS. 47A and 47B are cross sectional images of skin followingtransdermal delivery of a protein therapeutic across the skin. FIG. 47Ais a cross section of skin that was in contact with a transdermal devicedefining a nanotopography thereon, and FIG. 47B is a cross section ofskin that was in contact with a transdermal device including no patternof nanotopography formed thereon.

FIG. 48 graphically illustrates the blood serum concentration of aprotein therapeutic delivered with a device as described herein.

DETAILED DESCRIPTION OF REPRESENTATIVE EMBODIMENTS

Reference now will be made in detail to various embodiments of thedisclosed subject matter, one or more examples of which are set forthbelow. Each example is provided by way of explanation, not limitation.In fact, it will be apparent to those skilled in the art that variousmodifications and variations may be made in the present disclosurewithout departing from the scope or spirit of the subject matter. Forinstance, features illustrated or described as part of one embodimentmay be used on another embodiment to yield a still further embodiment.Thus, it is intended that the present disclosure covers suchmodifications and variations as come within the scope of the appendedclaims and their equivalents.

A medical device is disclosed herein that includes a pattern ofstructures fabricated on a surface, at least a portion of which arefabricated on a nanometer scale. As utilized herein, the term‘fabricated’ generally refers to a structure that has been specificallydesigned, engineered, and/or constructed so as to exist at a surface ofthe medical device and is not to be equated with a surface feature thatis merely an incidental product of the device formation process. Thus,there will be a predetermined pattern of nanostructures on the surfaceof the microneedles.

The medical device may be constructed from a variety of materials,including metals, ceramics, semiconductors, organics, polymers, etc., aswell as composites thereof. By way of example, pharmaceutical gradestainless steel, titanium, nickel, iron, gold, tin, chromium, copper,alloys of these or other metals, silicon, silicon dioxide, and polymersmay be utilized. Typically, the device is formed of a biocompatiblematerial that is capable of carrying a pattern of structures asdescribed herein on a surface. The term “biocompatible” generally refersto a material that does not substantially adversely affect the cells ortissues in the area where the device is to be delivered. It is alsointended that the material does not cause any substantially medicallyundesirable effect in any other areas of the living subject.Biocompatible materials may be synthetic or natural. Some examples ofsuitable biocompatible materials, which are also biodegradable, includepolymers of hydroxy acids such as lactic acid and glycolic acidpolylactide, polyglycolide, polylactide-co-glycolide, copolymers withpolyethylene glycol, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butyric acid), poly(valeric acid), andpoly(lactide-co-caprolactone). Other suitable materials may include,without limitation, polycarbonate, polymethacrylic acid, ethylenevinylacetate, polytetrafluorethylene, and polyesters. The device may likewisebe non-porous or porous in nature, may be homogeneous or heterogeneousacross the device with regard to materials, geometry, solidity, and soforth, and may have a rigid fixed or a semi-fixed shape.

Regardless of the materials employed, the medical device may be used forinteraction with tissue, such as in delivery of a bioactive agent to acell. For example, the medical device may be used to deliver an agent tothe tissue or to one or more cell types of the tissue, for structuralsupport of a tissue, for removal of a portion or component of thetissue, and so forth. The medical device may be used in one embodimentfor transport of a substance across one or more layers of the skin.During use, the device may interact with surrounding biologicalcomponents and regulate or modulate (i.e., change) intracellular and/orintercellular signal transduction associated with cell/cellinteractions, endocytosis, inflammatory response, and so forth. Forinstance, through interaction between the nanotopography on a surface ofthe medical device and surrounding biological materials or structures,the device may regulate and/or modulate membrane potential, membraneproteins, and/or intercellular junctions (e.g., tight junctions, gapjunctions, and/or desmasomes). The device may be utilized fortransdermal delivery of agents or withdrawal of materials withoutinstigating a foreign body or immune response.

In one embodiment, the device is a microneedle or a microneedle array,although it should be understood that the devices are not limited tomicroneedles. Microneedles may be useful in transport of material acrossbiological barriers such as the skin, the blood-brain barrier, mucosaltissues, blood and lymph vessels, and so forth. FIG. 1 illustrates atypical microneedle device 10. As may be seen, the device includes anarray of individual needles 12; each formed to a size and shape so as topenetrate a biological barrier without breakage of the individualmicroneedles. Microneedles may be solid, as in FIG. 1, porous, or mayinclude a hollow portion. A microneedle may include a hollow portion,e.g., an annular bore that may extend throughout all or a portion of theneedle, extending parallel to the direction of the needle or branchingor exiting at a side of the needle, as appropriate. For example, FIG. 2illustrates an array of microneedles 14 each including a channel 16 in aside of the needles as may be utilized for, e.g., delivery of an agentto a subdermal location. For instance, a channel 16 may be in at leastpartial alignment with an aperture in base 15 so as to form a junctionbetween the aperture and channel 16 allowing the passage of a substancethrough the channel 16.

The dimensions of the channel 16, when present, can be specificallyselected to induce capillary flow of a drug compound. Capillary flowgenerally occurs when the adhesive forces of a fluid to the walls of achannel are greater than the cohesive forces between the liquidmolecules. Specifically, capillary pressure is inversely proportional tothe cross-sectional dimension of the channel 16 and directlyproportional to the surface tension of the liquid, multiplied by thecosine of the contact angle of the fluid in contact with the materialforming the channel. Thus, to facilitate capillary flow in the patch,the cross-sectional dimension (e.g., width, diameter, etc.) of thechannel 16 may be selectively controlled, with smaller dimensionsgenerally resulting in higher capillary pressure. For example, in someembodiments, the cross-sectional dimension of the channel typicallyranges from about 1 micrometer to about 100 micrometers, in someembodiments from about 5 micrometers to about 50 micrometers, and insome embodiments, from about 10 micrometers to about 30 micrometers. Thedimension may be constant or it may vary as a function of the length ofthe channel 16. The length of the channel may also vary to accommodatedifferent volumes, flow rates, and dwell times for the drug compound.For example, the length of the channel may be from about 10 micrometersto about 800 micrometers, in some embodiments from about 50 micrometersto about 500 micrometers, and in some embodiments, from about 100micrometers to about 300 micrometers. The cross-sectional area of thechannel may also vary. For example, the cross-sectional area may be fromabout 50 square micrometers to about 1,000 square micrometers, in someembodiments from about 100 square micrometers to about 500 squaremicrometers, and in some embodiments, from about 150 square micrometersto about 350 square micrometers. Further, the aspect ratio(length/cross-sectional dimension) of the channel may range from about 1to about 50, in some embodiments from about 5 to about 40, and in someembodiments from about 10 to about 20. In cases where thecross-sectional dimension (e.g., width, diameter, etc.) and/or lengthvary as a function of length, the aspect ratio can be determined fromthe average dimensions.

It should be understood that the number of microneedles shown in thefigures is for illustrative purposes only. The actual number ofmicroneedles used in a microneedle assembly may, for example, range fromabout 500 to about 10,000, in some embodiments from about 2,000 to about8,000, and in some embodiments, from about 4,000 to about 6,000.

An individual microneedle may have a straight or a tapered shaft. In oneembodiment, the diameter of a microneedle may be greatest at the baseend of the microneedle and taper to a point at the end distal the base.A microneedle may also be fabricated to have a shaft that includes botha straight (untapered) portion and a tapered portion.

A microneedle may be formed with a shaft that is circular ornon-circular in cross-section. For example, the cross-section of amicroneedle may be polygonal (e.g. star-shaped, square, triangular),oblong, or any other shape. The shaft may have one or more bores and/orchannels.

The size of individual needles may be optimized depending upon thedesired targeting depth, the strength requirements of the needle toavoid breakage in a particular tissue type, etc. For instance, thecross-sectional dimension of a transdermal microneedle may be betweenabout 10 nanometers (nm) and 1 millimeter (mm), or between about 1micrometer (μm) and about 200 micrometers, or between about 10micrometers and about 100 micrometers. The outer diameter may be betweenabout 10 micrometers and about 100 micrometers and the inner diameter ofa hollow needle may be between about 3 micrometers and about 80micrometers. The tip typically has a radius that is less than or equalto about 1 micrometer.

The length of a microneedle will generally depend upon the desiredapplication. For instance, a microneedle may be between about 1micrometer and about 1 millimeter in length, for instance about 500micrometers or less, or between about 10 micrometers and about 500micrometers, or between about 30 micrometers and abut 200 micrometers.

An array of microneedles need not include microneedles that are allidentical to one another. An array may include a mixture of microneedleshaving various lengths, outer diameters, inner diameters,cross-sectional shapes, nanostructured surfaces, and/or spacings betweenthe microneedles. For example, the microneedles may be spaced apart in auniform manner, such as in a rectangular or square grid or in concentriccircles. The spacing may depend on numerous factors, including heightand width of the microneedles, as well as the amount and type of anysubstance that is intended to be moved through the microneedles. While avariety of arrangements of microneedles is useful, a particularly usefularrangement of microneedles is a “tip-to-tip” spacing betweenmicroneedles of about 50 micrometers or more, in some embodiments about100 to about 800 micrometers, and in some embodiments, from about 200 toabout 600 micrometers.

Referring again to FIG. 1, microneedles may be held on a substrate 20(i.e., attached to or unitary with a substrate) such that they areoriented perpendicular or at an angle to the substrate. In oneembodiment, the microneedles may be oriented perpendicular to thesubstrate and a larger density of microneedles per unit area ofsubstrate may be provided. However, an array of microneedles may includea mixture of microneedle orientations, heights, materials, or otherparameters. The substrate 20 may be constructed from a rigid or flexiblesheet of metal, ceramic, plastic or other material. The substrate 20 canvary in thickness to meet the needs of the device, such as about 1000micrometers or less, in some embodiments from about 1 to about 500micrometers, and in some embodiments, from about 10 to about 200micrometers.

According to the present disclosure, a microneedle surface may define ananotopography thereon in a random or organized pattern. FIG. 3schematically illustrates the ends of two representative microneedles22. Microneedles 22 define a central bore 24 as may be used for deliveryof an agent via the microneedles 22. The surface 25 of microneedles 22define nanotopography 26. In this particular embodiment, thenanotopography 26 defines a random pattern on the surface 25 of themicroneedle 22.

A microneedle may include a plurality of identical structures formed ona surface or may include different structures formed of various sizes,shapes and combinations thereof. A predetermined pattern of structuresmay include a mixture of structures having various lengths, diameters,cross-sectional shapes, and/or spacings between the structures. Forexample, the structures may be spaced apart in a uniform manner, such asin a rectangular or square grid or in concentric circles. In oneembodiment, structures may vary with regard to size and/or shape and mayform a complex nanotopography. For example, a complex nanotopography maydefine a fractal or fractal-like geometry.

As utilized herein, the term “fractal” generally refers to a geometricor physical structure having a fragmented shape at all scales ofmeasurement between a greatest and a smallest scale such that certainmathematical or physical properties of the structure behave as if thedimensions of the structure are greater than the spatial dimensions.Mathematical or physical properties of interest may include, forexample, the perimeter of a curve or the flow rate in a porous medium.The geometric shape of a fractal may be split into parts, each of whichdefines self-similarity. Additionally, a fractal has a recursivedefinition and has a fine structure at arbitrarily small scales.

As utilized herein, the term “fractal-like” generally refers to ageometric or physical structure having one or more, but not all, of thecharacteristics of a fractal. For instance, a fractal-like structure mayinclude a geometric shape that includes self-similar parts, but may notinclude a fine structure at an arbitrarily small scale. In anotherexample, a fractal-like geometric shape or physical structure may notdecrease (or increase) in scale equally between iterations of scale, asmay a fractal, though it will increase or decrease between recursiveiterations of a geometric shape of the pattern. A fractal-like patternmay be simpler than a fractal. For instance, it may be regular andrelatively easily described in traditional Euclidean geometric language,whereas a fractal may not.

A microneedle surface defining a complex nanotopography may includestructures of the same general shape (e.g., pillars) and the pillars maybe formed to different scales of measurement (e.g., nano-scale pillarsas well as micro-scale pillars). In another embodiment, a microneedlemay include at a surface structures that vary in both scale size andshape or that vary only in shape while formed to the same nano-sizedscale. Additionally, structures may be formed in an organized array orin a random distribution. In general, at least a portion of thestructures may be nanostructures formed on a nano-sized scale, e.g.,defining a cross-sectional dimension of less than about 500 nanometers,for instance less than about 400 nanometers, less than about 250nanometers, or less than about 100 nanometers. The cross sectionaldimension of the nanostructures can generally be greater than about 5nanometers, for instance greater than about 10 nanometers, or greaterthan about 20 nanometers. For example, the nanostructures can define across sectional dimension between about 5 nanometers and about 500nanometers, between about 20 nanometers and about 400 nanometers, orbetween about 100 nanometers and about 300 nanometers. In cases wherethe cross sectional dimension of a nanostructure varies as a function ofheight of the nanostructure, the cross sectional dimension can bedetermined as an average from the base to the tip of the nanostructures,or as the maximum cross sectional dimension of the structure, forexample the cross sectional dimension at the base of a cone-shapednanostructure.

FIG. 4 illustrates one embodiment of a complex nanotopography as may beformed on a surface. This particular pattern includes a central largepillar 100 and surrounding pillars 102, 104, of smaller dimensionsprovided in a regular pattern. As may be seen, this pattern includes aniteration of pillars, each of which is formed with the same generalshape, but vary with regard to horizontal dimension. This particularcomplex pattern is an example of a fractal-like pattern that does notinclude identical alteration in scale between successive recursiveiterations. For example, while the pillars 102 are first nanostructuresthat define a horizontal dimension that is about one third that of thelarger pillar 100, which is a microstructure, the pillars 104 are secondnanostructures that define a horizontal dimension that is about one halfthat of the pillars 102.

A pattern that includes structures of different sizes can include largerstructures having a cross-sectional dimension formed on a larger scale,e.g., microstructures having a cross-sectional dimension greater thanabout 500 nanometers in combination with smaller nanostructures. In oneembodiment, microstructures of a complex nanotopography can have across-sectional dimension between about 500 nanometers and about 10micrometers, between about 600 nanometers and about 1.5 micrometers, orbetween about 650 nanometers and about 1.2 micrometers. For example, thecomplex nanotopography of FIG. 4 includes micro-sized pillars 100 havinga cross sectional dimension of about 1.2 micrometers.

When a pattern includes one or more larger microstructures, forinstance, having a cross-sectional dimension greater than about 500nanometers, determined either as the average cross sectional dimensionof the structure or as the largest cross sectional dimension of thestructure, the complex nanotopography will also include nanostructures,e.g., first nanostructures, second nanostructures of a different sizeand/or shape, etc. For example, pillars 102 of the complexnanotopography of FIG. 4 have a cross-sectional dimension of about 400nanometers, and pillars 104 have a cross-sectional dimension of about200 nanometers.

A nanotopography can be formed of any number of different elements. Forinstance, a pattern of elements can include two different elements,three different elements, an example of which is illustrated in FIG. 4,four different elements, or more. The relative proportions of therecurrence of each different element can also vary. In one embodiment,the smallest elements of a pattern will be present in larger numbersthan the larger elements. For instance in the pattern of FIG. 4, thereare eight pillars 104 for each pillar 102, and there are eight pillars102 for the central large pillar 100. As elements increase in size,there can generally be fewer recurrences of the element in thenanotopography. By way of example, a first element that is about 0.5,for instance between about 0.3 and about 0.7 in cross-sectionaldimension as a second, larger element can be present in the topographyabout five times or more than the second element. A first element thatis approximately 0.25, or between about 0.15 and about 0.3 incross-sectional dimension as a second, larger element can be present inthe topography about 10 times or more than the second element.

The spacing of individual elements can also vary. For instance,center-to-center spacing of individual structures can be between about50 nanometers and about 1 micrometer, for instance between about 100nanometers and about 500 nanometers. For example, center-to-centerspacing between structures can be on a nano-sized scale. For instance,when considering the spacing of nano-sized structures, thecenter-to-center spacing of the structures can be less than about 500nanometers. This is not a requirement of a topography, however, andindividual structures can be farther apart. The center-to-center spacingof structures can vary depending upon the size of the structures. Forexample, the ratio of the average of the cross-sectional dimensions oftwo adjacent structures to the center-to-center spacing between thosetwo structures can be between about 1:1 (e.g., touching) and about 1:4,between about 1:1.5 and about 1:3.5, or between about 1:2 and about 1:3.For instance, the center to center spacing can be approximately doublethe average of the cross-sectional dimensions of two adjacentstructures. In one embodiment, two adjacent structures each having across-sectional dimension of about 200 nanometers can have acenter-to-center spacing of about 400 nanometers. Thus, the ratio of theaverage of the diameters to the center-to-center spacing in this case is1:2.

Structure spacing can be the same, i.e., equidistant, or can vary forstructures in a pattern. For instance, the smallest structures of apattern can be spaced apart by a first distance, and the spacing betweenthese smallest structures and a larger structure of the pattern orbetween two larger structures of the pattern can be the same ordifferent as this first distance.

For example, in the pattern of FIG. 4, the smallest structures 104 havea center-to-center spacing of about 200 nanometers. The distance betweenthe larger pillars 102 and each surrounding pillar 104 is less, about100 nanometers. The distance between the largest pillar 100 and eachsurrounding pillar 104 is also less than the center-to-center spacingbetween to smallest pillars 104, about 100 nanometers. Of course, thisis not a requirement, and all structures can be equidistant from oneanother or any variation in distances. In one embodiment, differentstructures can be in contact with one another, for instance atop oneanother, as discussed further below, or adjacent one another and incontact with one another.

Structures of a topography may all be formed to the same height,generally between about 10 nanometers and about 1 micrometer, but thisis not a requirement, and individual structures of a pattern may vary insize in one, two, or three dimensions. In one embodiment, some or all ofthe structures of a topography can have a height of less than about 20micrometers, less than about 10 micrometers, or less than about 1micrometer, for instance less than about 750 nanometers, less than about680 nanometers, or less than about 500 nanometers. For instance thestructures can have a height between about 50 nanometers and about 20micrometers or between about 100 nanometers and about 700 nanometers.For example, nanostructures or microstructures can have a height betweenabout 20 nm and about 500 nm, between about 30 nm and about 300 nm, orbetween about 100 nm and about 200 nm, though it should be understoodthat structures may be nano-sized in a cross sectional dimension and mayhave a height that may be measured on a micro-sized scale, for instancegreater than about 500 nm. Micro-sized structures can have a height thatis the same or different from nano-sized structures of the same pattern.For instance, micro-sized structures can have a height of between about500 nanometers and about 20 micrometers, or between about 1 micrometerand about 10 micrometers, in another embodiment. Micro-sized structuresmay also have a cross sectional dimension on a micro-scale greater thanabout 500 nm, and may have a height that is on a nano-sized scale ofless than about 500 nm.

The aspect ratio of the structures (the ratio of the height of astructure to the cross sectional dimension of the structure) can bebetween about 0.15 and about 30, between about 0.2 and about 5, betweenabout 0.5 and about 3.5, or between about 1 and about 2.5. For instance,the aspect ratio of the nanostructures may fall within these ranges.

The device surface may include a single instance of a pattern, as shownin FIG. 4, or may include multiple iterations of the same or differentpatterns. For example, FIG. 5 illustrates a surface pattern includingthe pattern of FIG. 4 in multiple iterations over a surface.

The formation of nanotopography on a surface may increase the surfacearea without a corresponding increase in volume. Increase in the surfacearea to volume ratio is believed to improve the interaction of a surfacewith surrounding biological materials. For instance, increase in thesurface area to volume ratio is believed to encourage mechanicalinteraction between the nanotopography and surrounding proteins, e.g.,extracellular matrix (ECM) proteins and/or plasma membrane proteins.

In general, the surface area to volume ratio of the device may begreater than about 10,000 cm⁻¹, greater than about 150,000 cm⁻¹, orgreater than about 750,000 cm⁻¹, Determination of the surface area tovolume ratio may be carried out according to any standard methodology asis known in the art. For instance, the specific surface area of asurface may be obtained by the physical gas adsorption method (B.E.T.method) with nitrogen as the adsorption gas, as is generally known inthe art and described by Brunauer, Emmet, and Teller (J. Amer. Chem.Soc., vol. 60, February, 1938, pp. 309-319), incorporated herein byreference. The BET surface area can be less than about 5 m²/g, in oneembodiment, for instance between about 0.1 m²/g and about 4.5 m²/g, orbetween about 0.5 m²/g and about 3.5 m²/g. Values for surface area andvolume may also be estimated from the geometry of molds used to form asurface, according to standard geometric calculations. For example, thevolume can be estimated according to the calculated volume for eachpattern element and the total number of pattern elements in a givenarea, e.g., over the surface of a single microneedle.

For a device that defines a complex pattern nanotopography at a surface,the nanotopography may be characterized through determination of thefractal dimension of the pattern. The fractal dimension is a statisticalquantity that gives an indication of how completely a fractal appears tofill space as the recursive iterations continue to smaller and smallerscale. The fractal dimension of a two dimensional structure may berepresented as:

$D = \frac{\log \; {N(e)}}{\log (e)}$

where N(e) is the number of self-similar structures needed to cover thewhole object when the object is reduced by 1/e in each spatialdirection.

For example, when considering the 2 dimensional fractal known as theSierpenski triangle illustrated in FIG. 6, in which the mid-points ofthe three sides of an equilateral triangle are connected and theresulting inner triangle is removed, the fractal dimension is calculatedas follows:

$D = \frac{\log \; {N(e)}}{\log (e)}$$D = \frac{\log \; 3}{\log \; 2}$ D ≈ 1.585

Thus, the Sierpenski triangle fractal exhibits an increase in linelength over the initial two dimensional equilateral triangle.Additionally, this increase in line length is not accompanied by acorresponding increase in area.

The fractal dimension of the pattern illustrated in FIG. 4 isapproximately 1.84. In one embodiment, nanotopography of a surface ofthe device may exhibit a fractal dimension of greater than about 1, forinstance between about 1.2 and about 5, between about 1.5 and about 3,or between about 1.5 and about 2.5.

FIGS. 7A and 7B illustrate increasing magnification images of anotherexample of a complex nanotopography. The nanotopography of FIGS. 7A and7B includes an array of fibrous-like pillars 70 located on a substrate.At the distal end of each individual pillar, the pillar splits intomultiple smaller fibers 60. At the distal end of each of these smallerfibers 60, each fiber splits again into multiple filaments (not visiblein FIGS. 7A and 7B). Structures formed on a surface that have an aspectratio greater than about 1 may be flexible, as are the structuresillustrated in FIGS. 7A and 7B, or may be stiff.

FIGS. 7C and 7D illustrate another example of a complex nanotopography.In this embodiment, a plurality of pillars 72 each including an annularhollow therethrough 71 are formed on a substrate. At the distal end ofeach hollow pillar, a plurality of smaller pillars 62 is formed. As maybe seen, the pillars of FIGS. 7C and 7D maintain their stiffness andupright orientation. Additionally, and in contrast to previous patterns,the smaller pillars 62 of this embodiment differ in shape from thelarger pillars 72. Specifically, the smaller pillars 62 are not hollow,but are solid. Thus, nanotopography including structures formed to adifferent scale need not have all structures formed with the same shape,and structures may vary in both size and shape from the structures of adifferent scale.

FIG. 8 illustrates another pattern including nano-sized structures asmay be formed on the device surface. As may be seen, in this embodiment,individual pattern structures may be formed at the same general size,but with different orientations and shapes from one another.

In addition to or alternative to those methods mentioned above, asurface may be characterized by other methods including, withoutlimitation, surface roughness, elastic modulus, and surface energy.

Methods for determining the surface roughness are generally known in theart. For instance, an atomic force microscope process in contact ornon-contact mode may be utilized according to standard practice todetermine the surface roughness of a material. Surface roughness thatmay be utilized to characterize a microneedle can include the averageroughness (R_(A)), the root mean square roughness, the skewness, and/orthe kurtosis. In general, the average surface roughness (i.e., thearithmetical mean height of the surface are roughness parameter asdefined in the ISO 25178 series) of a surface defining a fabricatednanotopography thereon may be less than about 200 nanometers, less thanabout 190 nanometers, less than about 100 nanometers, or less than about50 nanometers. For instance, the average surface roughness may bebetween about 10 nanometers and about 200 nanometers, or between about50 nanometers and about 190 nanometers.

The device may be characterized by the elastic modulus of thenanopatterned surface, for instance by the change in elastic modulusupon the addition of a nanotopography to a surface. In general, theaddition of a plurality of structures forming nanotopography on asurface can decrease the elastic modulus of a material, as the additionof nano-sized structures on a surface will lead to a reduction incontinuity of the surface and a related change in surface area. Ascompared to a similar surface formed according to the same process andof the same materials, but for a pattern of nanotopography on thesurface, the device including nanotopography thereon can exhibit adecrease in elastic modulus of between about 35% and about 99%, forinstance between about 50% and about 99%, or between about 75% and about80%. By way of example, the effective compression modulus of ananopatterned surface can be less than about 50 MPa, or less than about20 MPa. In one embodiment the effective compression modulus can bebetween about 0.2 MPa and about 50 MPa, between about 5 MPa and about 35MPa, or between about 10 MPa and about 20 MPa. The effective shearmodulus can be less than about 320 MPa, or less than about 220 MPa. Forinstance, the effective shear modulus can be between about 4 MPa andabout 320 MPa, or between about 50 MPa and about 250 MPa, in oneembodiment.

The device including nanotopography thereon may also exhibit an increasein surface energy as compared to a similar microneedle that does nothave a surface defining a pattern of nanotopography thereon. Forinstance, a microneedle including a nanotopography formed thereon canexhibit an increase in surface energy as compared to a similarmicroneedle of the same materials and formed according to the samemethods, but for the inclusion of a pattern of nanotopography on asurface. For instance, the water contact angle of a surface including ananotopography thereon can be greater than about 80°, greater than about90°, greater than about 100°, or greater than about 110°. For example,the water contact angle of a surface can be between about 80° and about150°, between about 90° and about 130°, or between about 100° and about120°, in one embodiment.

When forming nanostructures on the surface of the device, the packingdensity of the structures may be maximized. For instance, square packing(FIG. 9A), hexagonal packing (FIG. 9B), or some variation thereof may beutilized to pattern the elements on a substrate. When designing apattern in which various sized elements of cross sectional areas A, B,and C are adjacent to one another on a substrate, circle packing asindicated in FIG. 9C may be utilized. Of course, variations in packingdensity and determination of associated alterations in characteristicsof a surface are well within the abilities of one of skill in the art.

During use, a microneedle device may interact with one or morecomponents of the dermal connective tissue. Connective tissue is theframework upon which the other types of tissue, i.e., epithelial,muscle, and nervous tissues, are supported. Connective tissue generallyincludes individual cells held within the ECM. The ECM, in turn,includes the ground substance (e.g., the minerals of bone, the plasma ofblood, etc.) and the fibrous component including collagen, fibronectin,laminins, etc. Connective tissue may assume widely divergentarchitectures, ranging from blood, in which the fibrous component isabsent and the ground substance is fluid, to dense connective tissue asis found in the skin, which includes a relatively high proportion ofextracellular fibers (e.g., collagen) and may contain little of theother connective tissue components. There are many specialized types ofconnective tissue in skin, one example being elastic tissue, in whichelastic fibers are the major component of the tissue and the amount offactors commonly found in other types of connective tissue, such ascollagen and proteoglycans, may be minimal.

The nanotopography of a microneedle surface may provide improvedinteraction between the microneedle and biological components of thedermal connective tissue of the delivery area. For instance,microneedles of a transdermal device may interact directly with ECMproteins and/or individual cells such as keratinocytes, Langerhans cellsof the stratum spinosum or the undifferentiated basal cells of thestratum germinativum. Longer needles on transdermal devices may beutilized to access components of the dermis, for instance blood cells ofthe capillary bed. Due to the improved interaction between the deviceand local biological components, the surrounding tissue may be lesslikely to exhibit a foreign body response, which may decrease localinflammation and improve delivery of active agents. In one embodiment,the device can play a more active roll in agent delivery. For instance,interaction between a nanotopography and the surrounding biologicalcomponents can encourage delivery of high molecular weight materials,for instance through opening of tight junctions in the stratumgranulosum.

While not wishing to be held to any particular theory, it is believedthat the nanotopography facilitates improved interaction with biologicalcomponents through two mechanisms. According to one mechanism, ananotopography may facilitate the ability of a microneedle to mimic theECM at a delivery site. For instance, the nanotopography of amicroneedle can mimic one or more components of the basement membrane ata delivery site. In use, a cell may contact the nanotopography of amicroneedle and react in a similar fashion to typical contact with thenatural structure (e.g., the basement membrane protein) that thenanotopography mimics. Accordingly, the device may directly interactwith a cell to regulate or modulate (i.e., change) cell behavior, e.g.,cell signal transduction, thereby improving delivery of an agent acrossnatural barriers as well as improving endocytosis of agents delivered bythe device.

According to a second mechanism, a nanotopography may interact withnon-cellular biological components of the local connective tissue suchas ECM proteins. For instance, ECM proteins may be adsorbed and desorbedfrom the surface of a microneedle. The adsorption/desorption of ECMproteins may alter the chemistry of the local environment, which canlead to alterations in cell behavior. According to this secondmechanism, the device may indirectly affect the behavior of a cell. Forexample, the adsorption of one or more ECM proteins at the surface ofthe device can indirectly regulate or modulate intracellular and/orintercellular signal transduction.

Due to improved interaction with surrounding biological components, thedevices can facilitate improved uptake of a delivered agent. Forexample, the pharmacokinetic (PK) profile (i.e., the profile ofabsorption through the epithelial membranes) of a protein therapeuticcan be enhanced through utilization of a device including a pattern ofnanotopography. By way of example, a protein therapeutic having amolecular weight of over 100 kDa, for instance between about 100 kDa andabout 200 kDa, or about 150 kDa, can be delivered transdermally via apatch defining a nanotopography thereon. In one embodiment, a patch canbe utilized to deliver a single dose of the protein therapeutic, forinstance between about 200 and about 500 μL, or about 250 μL. Followingattachment of the transdermal patch to the skin, the recipient canexhibit a PK profile that reflects a rapid rise in blood serumconcentration up to between about 500 and about 1000 nanogramstherapeutic per milliliter per square centimeter of patch area, forinstance between about 750 and about 850 nanograms therapeutic permilliliter per square centimeter patch area, within about 1 to about 4hours of administration. This initial rapid rise in blood serum level,which reflects rapid uptake of the therapeutic across the dermalbarrier, can be followed by a less rapid decline of blood serumconcentration over between about 20 and about 30 hours, for instanceover about 24 hours, down to a negligible blood serum concentration ofthe therapeutic. Moreover, the rapid uptake of the delivered therapeuticcan be accompanied by little or no inflammation. Specifically, inaddition to promoting improved delivery of an agent across a transdermalbarrier, the devices can also limit foreign body response and otherundesirable reactions, such as inflammation. Use of previously knowndevices, such as transdermal patches with no nanotopography defined atthe skin contacting surface, often led to local areas of inflammationand irritation.

Structures of the nanotopography may mimic and/or interact with one ormore ECM protein such as collagen, laminin, fibronectin, etc. This maydirectly or indirectly alter a cell membrane protein with regard to oneor more characteristics such as conformation, free energy, localdensity. Exemplary cell membrane proteins include, without limitation,integrins, viniculin or other focal adhesion proteins, clathrin,membrane receptors such as G protein coupled receptors, etc. Thisalteration may induce changes at the cell surface and/or within the cellvia downstream effects through the cytoskeleton and within thecytoplasm.

Cells in the local area surrounding the device may maintain ananti-inflammatory microenvironment as the device may better mimic thelocal environment either directly or indirectly due to proteinadsorption at the surface. Thus, materials may be delivered by use ofthe device without development of a foreign body or immune response.

Specific cell types that may be directly or indirectly affected by thepresence of a microneedle may include cells of the surrounding dermalconnective tissue. For instance, a microneedle surface definingnanotopography may be located in an area that includes Langerhans cells,macrophages, and/or T-cells without triggering a foreign body or immuneresponse. Langerhans cells may take up and process an antigen to becomea fully functional antigen presenting cell. Macrophages and T-cells playa central role in the initiation and maintenance of the immune response.Once activated by pathological or immunogenic stimuli, for instance viaa Langerhans cell, T-cells may release IL-2, IL-4, INF-γ, and otherinflammatory cytokines. Macrophages respond in the process by releasinga host of inflammatory mediators, including TNF-α, IL-1, IL-8, IL-11,IL-12, nitric oxide, IL-6, GM-CSF, G-CSF, M-CSF, IFN-α, IFN-β andothers. Released cytokines activate other immune cells and some may alsoact as independent cytotoxic agents. Excessive release of macrophage andT-cell derived inflammatory mediators may lead to damage of normal cellsand surrounding tissues.

Without wishing to be bound to any particular theory, it is believedthat through interaction with a nanopatterned substrate, individualcells can up- or down-regulate the production of certain cytokines,including certain chemokines. Through that alteration in expressionprofile, cellular response to a drug delivery device can be minimized.For example, inflammation and/or foreign body response can be minimizedthrough upregulation of one or more anti-inflammatory cytokines and/ordown-regulation of one or more pro-inflammatory cytokines. Manycytokines have been characterized according to effect on inflammation.Pro-inflammatory cytokines that may demonstrate altered expressionprofiles when expressing cells are affected by the presence of a deviceincluding a nanotopography fabricated thereon can include, withoutlimitation, IL-1α, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, IL16, MIG,MIP-1α, MIP-1β, KC, MCP-1, TNF-α, GM-CSI, VEGF, and the like.Anti-inflammatory cytokines that may demonstrate an altered expressionprofile can include, without limitation, IL-1ra, IL-4, IL-10, IL-13, andthe like. Cytokines associated with foreign body response that candemonstrate an altered expression profile can include, withoutlimitation, IL-4, IL-10, IL-13, and so forth.

Nitric oxide is recognized as a mediator and regulator of inflammatoryresponses. By influencing the local environment, a microneedle may limitthe release of nitric oxide from surrounding cells. This may bebeneficial as nitric oxide may possess toxic properties toward an activeagent being delivered and may also have deleterious effects on thesubject's own tissue (Korhonen et al., Curr Drug Targets Inflamm Allergy4(4): 471-9, 2005). Nitric oxide may also interact with molecular oxygenand superoxide anion to produce reactive oxygen species (ROS) that maymodify various cellular functions. These indirect effects of nitricoxide have a significant role in inflammation; in which nitric oxide maybe produced in high amounts by inducible nitric oxide synthase (iNOS)and ROS may be synthesized by activated inflammatory cells.

Nitric oxide may be produced by keratinocytes, fibroblasts, endothelialcells, and possibly others, any of which may be directly or indirectlyaffected by the nanotopography of a microneedle. Inhibition of nitricoxide synthesis as may be provided by the nanotopography of amicroneedle surface may affect wound contraction, alter collagenorganization, and alter neoepidermis thickness. Mast cell migration andangiogenesis in wounds may also be affected by inhibition of nitricoxide. Due to variable pathways of regulation, and without being boundto any particular theory, the device may increase nitric oxideproduction and/or retard nitric oxide degradation, whereas in anotherembodiment, the device may decrease nitric oxide production and/orhasten nitric oxide degradation.

Interaction of the device with components of a cell network or layer ofthe epidermis may modulate (i.e., change) the structure of intercellularjunctions therein. An intracellular junction may be at least onejunction selected from the group consisting of tight junctions, gapjunctions, and desmasomes. By way of example, interaction betweenbiological components and structures of a nanotopography may modulateproteins of a cellular network so as to induce the opening of tightjunctions of the stratum granulosum, thereby providing improved deliveryof an active agent across the epidermis, and in one particularembodiment, a high molecular weight active agent.

The nanotopography of the device may mimic and/or adsorb one or morecomponents of the ECM. In general, the ECM includes both theinterstitial matrix and the basement membrane. The interstitial matrixis composed of complex mixtures of proteins and proteoglycans and, inthe case of bone, mineral deposits. The basement membrane includes bothbasal lamina and reticular lamina and anchors and supports theepithelium and the endothelium. The specific make-up of the ECM may varydepending upon the specific tissue type, but in general will include avariety of collagens, laminins, fibronectin, and elastins. Thus, thenanotopography of the device may be designed to interact with componentsof a specific location or alternatively may be more generally designed,e.g., to interact with components of the dermal structure common to mostskin.

Structures of the nanotopography can interact with collagen, which is acommon basement membrane protein found in dermal ECM. Collagens areinsoluble, extracellular glycoproteins that are found in all animals andare the most abundant proteins in the human body. They are essentialstructural components of most connective tissues, including cartilage,bone, tendons, ligaments, fascia and skin. To date, 19 types ofcollagens have been found in humans. The major types include Type I,which is the chief component of tendons, ligaments, and bones; Type II,which represents more than 50% of the protein in cartilage, and is alsoused to build the notochord of vertebrate embryos; Type III, whichstrengthens the walls of hollow structures like arteries, the intestine,and the uterus, and Type IV, which forms the basal lamina of epithelia.A meshwork of Type IV collagens provides the filter for the bloodcapillaries and the glomeruli of the kidneys. The other 15 types ofcollagen, while being much less abundant, are no less important to thefunction of the ECM.

The basic unit of collagens is a polypeptide that often follows thepattern Gly-Pro-Y or Gly-X-Hyp, where X and Y may be any of variousother amino acid residues. The resulting polypeptide is twisted into anelongated, left-handed helix. When synthesized, the N-terminal andC-terminal of the polypeptide have globular domains, which keep themolecule soluble.

As utilized herein, the term “polypeptide” generally refers to amolecular chain of amino acids and does not refer to a specific lengthof the product. Thus, peptides, oligopeptides and proteins are includedwithin the definition of polypeptide. This term is also intended toinclude polypeptides that have been subjected to post-expressionmodifications such as, for example, glycosylations, acetylations,phosphorylations and so forth. As utilized herein, the term “protein”generally refers to a molecular chain of amino acids that is capable ofinteracting structurally, enzymatically or otherwise with otherproteins, polypeptides or any other organic or inorganic molecule.

Common amino acid symbol abbreviations as described below in Table 1 areused throughout this disclosure.

TABLE 1 Amino Acid One letter symbol Abbreviation Alanine A Ala ArginineR Arg Asparagine N Asn Aspartic acid D Asp Cysteine C Cys Glutamine QGln Glutamic acid E Glu Glycine G Gly Histidine H His Isoleucine I IleLeucine L Leu Lysine K Lys Methionine M Met Phenylalanine F Phe ProlineP Pro Serine S Ser Threonine T Thr Tryptophan W Trp Tyrosine Y TyrValine V Val

Tropocollagen is a subunit of larger collagen aggregates such asfibrils. It is approximately 300 nanometers long and 1.5 nanometers indiameter, made up of three polypeptide strands, each possessing theleft-handed helix conformation.

Nanotopography of the device surface can interact with and/or mimictropocollagen as well as collagen. In one embodiment, a nanotopographymay be more complex and can mimic and/or interact with bothtropocollagen on a nanoscale and collagen on a microscale. For instance,a larger component of a nanotopography can mimic the three left-handedhelices of tropocollagen that are twisted together into a right-handedcoiled coil, a triple helix or “super helix”, a cooperative quaternarystructure stabilized by numerous hydrogen bonds. With type I collagenand possibly all fibrillar collagens if not all collagens, eachtriple-helix associates into a right-handed super-super-coil that isreferred to as the collagen microfibril. Each microfibril isinterdigitated with its neighboring microfibrils. In some collagens(e.g., Type II), the three polypeptides that make up the microfibril areidentical. In other collagens (e.g., Type I), two polypeptides of onekind (gene product) assemble with a second similar but differentpolypeptide.

Laminin is another common basement membrane protein of the skin that maybe found in the local area of the device. Laminin is one of a family ofheterotrimer protein complexes formed from various combinations ofdifferent α, β, and γ subunit chains. Laminins in general may be foundprimarily in the basement membranes of the ECM and interact with othermatrix macromolecules to contribute to cell differentiation, movementand maintenance. The different laminin chains, α-1 through α-5, β-1through β-3, and γ-1 through γ-3 may theoretically form many differenttrimeric isoforms, but the existence of only 15 of the possible isoformshas been confirmed.

Laminins are in the shape of a cross including three shorter arms andone long arm. The three shorter arms are particularly good at binding toother laminin molecules, leading to formation of sheets in the basementmembrane. The long arm generally is the cell binding locale, binding tocell membranes and other ECM molecules, which helps anchor organizedtissue cells to the membrane.

Laminins further include subdomains with specific geometries. Forinstance, the terminal portion of the laminin-332 α3 chain, the Gdomain, is further subdivided into 5 sub-domains, G1, G2, G3, G4, andG5. The G subdomains of the laminin-332 α3 chain have been shown to benecessary for adherence of laminin-332 to cells which have certainreceptor integrins on their cell surface. Accordingly, nano-sizedstructures of a nanotopography may mimic one or more subdomains oflaminin. In a more complex pattern, these nano-sized structures may becombined with larger structures that may mimic an entire lamininprotein. A nanotopography may also or alternatively adsorb/desorblaminin and thereby affect the local environment.

A nanotopography may interact with fibronectin, which is involved intissue repair, embryogenesis, blood clotting and cell migration andadhesion. In the ECM fibronectin exists as an insoluble glycoproteindimer. The structure of fibronectin is rod-like, composed of threedifferent types of homologous, repeating modules, Types I, II, and III.These modules, though all part of the same amino acid chain, aretypically envisioned as “beads on a string,” each one joined to itsneighbors by short linkers.

Twelve Type I modules make up the amino-terminal and carboxy-terminalregion of the protein, and are involved mainly in fibrin and collagenbinding. Only two type II modules are found in fibronectin. They areinstrumental in binding collagen. The most abundant module infibronectin is Type III, which contains the RGD fibronectin receptorrecognition sequence along with binding sites for other integrins andheparin. Depending on the tissue type and/or cellular conditions, thefibronectin molecule is made up of 15-17 type III modules. In addition,there is a module that does not fall into any of these categories,called IIICS. This module, along with EDB and EDA (both type IIImodules), is regulated through alternative splicing of FN pre-mRNA.Fibronectin molecules may form two disulphide bridges at theircarboxy-termini, producing a covalently-linked dimer. A cell in contactwith the nanotopography of a microneedle device may interact with thedevice in similar fashion to the normal interaction of the cell withfibronectin.

Yet another common ECM protein that a device can interact with iselastin and/or a polypeptide fragment of elastin. Elastin is the proteinconstituent of connective tissue responsible for the elasticity andrecoil of the tissue. Moreover, elastin is quite abundant in connectivetissue. Tropoelastin chains are naturally cross-linked together to formelastic fibers. Unlike collagen, elastin molecules may uncoil into amore extended conformation when the fiber is stretched and will recoilspontaneously as soon as the stretching force is relaxed. Elastin isprimarily composed of Gly, Val, Ala, and Pro. It defines an irregular orrandom coil conformation.

In addition to the common dermal fibrous proteins, the nanotopography ofthe device may mimic and/or adsorb other ECM components such asproteoglycans. Proteoglycans are glycoproteins but consist of much morecarbohydrate than protein; that is, they are huge clusters ofcarbohydrate chains often attached to a protein backbone. Several sugarsare incorporated in proteoglycans. The most abundant one isN-acetylglucosamine (NAG). The long chains of sugar residues areattached to serine residues in the protein backbone; that is, they are“O-linked”. Sulfate groups are also added to the sugars prior tosecretion. Examples of common ECM proteoglycans include, withoutlimitation, chondroitin sulfate, heparan sulfate, keratan sulfate, andhyaluronic acid (which has no protein component.

A nanotopography on a surface may directly and/or indirectly affect acell in the local area of the device. This may include a cell of abarrier layer that lies between the skin surface and the delivery siteof an agent to be delivered by a microneedle device as well as a cell towhich an agent is to be delivered. Specific affects on a cell due to thepresence of the device may include alteration of a conformation, ligandbinding activity, or a catalytic activity of a membrane associatedprotein.

Presence of the device may modulate cellular membrane conductivity,including, in one aspect, whole-cell conductance. Moreover, modulatingwhole-cell conductance may include modulating at least one of linear andnon-linear voltage-dependent contribution of the whole-cell conductance.The device may modulate at least one of cellular membrane potential andcellular membrane conductivity. For instance, the device may directly orindirectly affect a calcium dependant cellular messaging pathway orsystem.

The nanotopography of the device may affect a component of a plasmamembrane, which may affect signaling pathways that are downstreameffectors of transcription factors. For instance, through mimicking orinteracting with a component of the ECM, the device may affect genetictranscription and/or translation within a local cell. Presence of thedevice may affect location and/or conformation of membrane proteins.This may in turn affect free energy of the local environment, leading toencouragement of endocytosis of an active agent delivered by the device.Presence of the device may affect the formation of junctions betweencells, e.g., tight junctions, leading to improved delivery of agentsacross a biological barrier.

It is believed that the device may directly or indirectly affect a cellvia a membrane associated protein. Membrane associated proteins mayinclude at least one of, without limitation, surface receptors,transmembrane receptors, ion channel proteins, intracellular attachmentproteins, cellular adhesion proteins, integrins, etc. According tocertain aspects, a transmembrane receptor may be a G-Protein CoupledReceptor (GPCR), For instance, the device may mimic an ECM componentthat interacts with a GPCR that in turn interacts with a G protein αsubunit. A G protein α subunit may be any of a Gα_(s), Gα_(i), Gα_(q),and Gα₁₂. In interacting with a component of the ECM, the device mayaffect cadherins, focal adhesions, desmosomes, integrins, clathrin,caveolin, TSLP receptor, β-2 adrenergic receptor, bradykinin receptor,ion channel proteins, and so forth. In one embodiment, presence of amicroneedle may modulate junction adhesion molecules including, withoutlimitation, JAM 2 and 3, GJA1, 3, 4 and 5 (junctional adherins),occludin (OCLN), claudins (e.g., CLDN 3, 5, 7, 8, 9, 10), and tightjunction protein 1 (TJP1).

The device may influence cellular activity not only at the cell surface,but internally as well. For instance, the device may influence a focaladhesion. Focal adhesions are large assemblies of materials that mayinclude 100 or more different proteins at any one time. They are dynamicin most cell types and provide a route for the transmission ofinformation, both mechanical and chemical, from the ECM to the innercell. Modification of focal adhesions takes place upon changes in themolecular composition, the physical structure, as well as physicalforces present in the ECM.

Focal adhesions allow connection between the cytoskeleton and the ECMand are generally considered to be a signaling hub for both mechanicalforce and chemical signal transmission. The bulk of focal adhesions arebeneath the cellular membrane, with connection to the ECM generally viaintegrins, though communication may also be via other transmembranematerials including hyaluronan and heparin-sulfate binding proteins.

Integrins are a large family of obligate heterodimeric transmembraneglycoproteins that attach cells to ECM proteins (e.g., laminin) of thebasement membrane or to ligands on other cells. The nanotopography ofthe device can affect integrin at the plasma membrane to affect the cellbehavior, for instance via a focal adhesion. Integrins contain large (a)and small (13) subunits of sizes 120-170 kDa and 90-100 kDa,respectively. In mammals, 18 α and β subunits have been characterized.Some integrins mediate direct cell to cell recognition and interactions.Integrins contain binding sites for divalent cations Mg²⁺ and Ca²⁺,which are necessary for their adhesive function. Mammalian integrinsform several subfamilies sharing common β subunits that associate withdifferent α subunits. Both the α and β subunits contain two separatetails, both of which penetrate the plasma membrane and possess smallcytoplasmic domains. The exception is the β-4 subunit which has acytoplasmic domain of 1088 amino acids, one of the largest knowncytoplasmic domains of any membrane protein. The penetrating portionsmay interact with proteins within a focal adhesion to communicateinformation with regard to the ECM to the cytoskeleton and the innercell. Outside the plasma membrane, the α and β chains lie close togetheralong a length of about 23 nanometers, with the final 5 nanometersN-termini of each chain forming a ligand-binding region for the ECM.

Primary proteins known within focal adhesions that may be affected bythe presence of a nanotopography in the area of the cell surface includevinculin, paxilin, talin, α-actinin, and zyxin. The focal adhesionproteins transmit information to the cytoskeleton, for instance viainteraction with actin, and throughout the cytoplasm. Focal adhesionsare in a constant state of flux, however, and proteins are continuallyassociating and disassociating with the complex, relating informationfrom the ECM to other parts of the cell. The dynamic assembly anddisassembly of focal adhesions from the formation of focal complexes atthe leading edge of the lamellipodia to the dissolution of the focaladhesion at the trailing edge of the cell is a central role in cellmigration. The activation of Src kinase due to extracellular mechanicalforces exerted on the inner cell via focal adhesion is an indication ofthe role focal adhesions play in sensing of mechanical forces of theECM. Accordingly, the device may modulate activity internal to the cell,including downstream functions involved with cell migration via plasmamembrane proteins associated with focal adhesions.

Other cell surface structures that nanotopography of the device mayaffect include membrane proteins involved in endocytosis. For instance,upon interaction with the cell membrane, the device may exhibitincreased adhesion energy due to the nanotopography of the contactingsurface. The adhesion energy may mimic that of typical receptor-ligandbinding.

While not wishing to be bound to any particular theory, it is believedthat upon adhesion between the surface of the device and an endocytosismediating receptor, other endocytosis mediating receptors in the cellmembrane may diffuse from a pre-adhesion uniform distribution on thecell membrane to the adhesion site. These membrane proteins may thenadhere to the device surface and thereby lower the free energy ofinteraction between the cell surface and the device. The lower freeenergy may encourage endocytosis of agents at the cell surface, e.g.,active agents delivered via the device. Specifically, when adhesionoccurs between the device surface and a receptor, the released energy,i.e., the increased free energy, may drive wrapping of the membranearound particles at or near the adhesion site. Alternatively, adsorptionof non-cellular components of the ECM to the surface of the device mayalter the local chemistry, leading to an increase in endocytic activityby cells in the area. Endocytosis pathways as may be mediated due to thepresence of the device may be subdivided into four categories includingclathrin-mediated endocytosis, caveolae, macropinocytosis, andphagocytosis.

Clathrin-mediated endocytosis is mediated by small vesicles about 100nanometers in diameter that have a morphologically characteristiccrystalline coat of a complex of proteins that mainly associate with thecytosolic protein clathrin. These clathrin-coated vesicles (CCVs) arefound in virtually all cells and form clathrin-coated pits on the plasmamembrane. Clathrin-coated pits may include increased concentration oflarge extracellular molecules that have different receptors responsiblefor the receptor-mediated endocytosis of ligands, e.g. low densitylipoprotein, transferrin, growth factors, antibodies and many others.

Nanotopography of the device may affect membrane proteins associatedwith caveolae. Caveolae are membrane endocytosis formations that areonly slightly less common than clathrin-coated pits, and exist on thesurface of many, but not all cell types. They consist of thecholesterol-binding protein caveolin (Vip21) with a bilayer enriched incholesterol and glycolipids. Caveolae are smaller than CCVs, about 50nanometers in diameter, and form flask-shape pits in the membrane. Theymay constitute up to a third of the plasma membrane area of the cells ofsome tissues, being especially abundant in fibroblasts, adipocytes, andendothelial cells as may be found in the skin. Uptake of extracellularmolecules is believed to be specifically mediated via receptors incaveolae as may be affected by nanotopography of a microneedle.

Macropinocytosis, which usually occurs from highly ruffled regions ofthe plasma membrane, describes the pulling in of the cell membrane toform a pocket, which then pinches off into the cell to form a largevesicle (0.5-5 μm in diameter) filled with large volume of extracellularfluid and molecules within it (equivalent to 103 to 106 CCVs). Thefilling of the pocket occurs in a non-specific manner. The vesicle thentravels into the cytosol and fuses with other vesicles such as endosomesand lysosomes.

Phagocytosis describes the binding and internalization of particulatematter larger than around 0.75 μm in diameter, such as small-sized dustparticles, cell debris, micro-organisms and even apoptotic cells, whichonly occurs in specialized cells. These processes involve the uptake oflarger membrane areas than clathrin-mediated endocytosis and thecaveolae pathway.

Nanotopography of the device may also affect cadherins of a cellsurface. Cadherins are a family of receptors generally involved inmediation of calcium-dependent homophilic cell-cell adhesion. Cadherinsare of primary importance during embryogenesis, but also play a role informing stable cell to cell junctions and maintaining normal tissuestructure in adults. Cadherins are a superfamily of transmembraneproteins grouped by the presence of one or more cadherin repeats intheir extracellular domains. Arrays of these approximately 110 residuedomains form the intermolecular surfaces responsible for the formationof cadherin-mediated cell-cell interactions. Structural information fromthe analysis of several cadherin domains indicates that calcium ionsbind at sites between adjacent cadherin repeats (CRs), forming a rigidrod. Cadherins typically include five tandem repeated extracellulardomains, a single membrane-spanning segment and a cytoplasmic region.

In one embodiment, the device may affect E-cadherin that is found inepithelial tissue. E-cadherin includes 5 cadherin repeats (EC1˜EC5) inthe extracellular domain, one transmembrane domain, and an intracellulardomain that binds p120-catenin and β-catenin. The intracellular domaincontains a highly-phosphorylated region that is believed to be vital toβ-catenin binding and therefore to E-cadherin function. β-catenin mayalso bind to α-catenin, and α-catenin participates in regulation ofactin-containing cytoskeletal filaments. In epithelial cells,E-cadherin-containing cell-to-cell junctions are often adjacent toactin-containing filaments of the cytoskeleton.

Other activities that may be affected due to the presence of the devicein a local area include paracellular and transcellular transport. Forinstance, through adsorption of ECM proteins at the surface of thedevice, the local chemistry of an area can change, leading to improvedparacellular transport of a delivered agent to the local area, which caninclude not only the immediate area of the device, but also areas deeperin the dermis. For instance, presence of the device can encourageparacellular transport of a delivered agent to the capillary beds of thedermis and across the capillary wall, so as to be delivered to the bloodstream for systemic delivery.

During use, the device may interact with one or more components of thecontacting epithelial tissue to increase porosity of the tissue viaparacellular and/or transcellular transport mechanisms. Epithelialtissue is one of the primary tissue types of the body. Epithelial tissueas may be rendered more porous according to the present disclosure caninclude both simple and stratified epithelium, including bothkeratinized epithelium and transitional epithelium. In addition,epithelial tissue encompassed herein can include any cell types of anepithelial layer including, without limitation, keratinocytes, squamouscells, columnar cells, cuboidal cells and pseudostratified cells.

Presence of the device may affect formation and maintenance of cell/celljunctions including tight junctions and desmosomes to encourageparacellular transport. As previously mentioned, tight junctions havebeen found in the stratum granulosum and opening of the tight junctionsmay provide a paracellular route for improved delivery of active agents,particularly large molecular weight active agents and/or agents thatexhibit low lipophilicity that have previously been blocked fromtransdermal delivery. The major types of proteins of tight junctions areclaudins, occludins, and junctional adhesion molecules.

Interaction between a local component and structures of thenanotopography may open desmosomes in a barrier layer to encourageparacellular transport. Desmosomes are formed primarily of desmogleinand desmocollin, both members of the cadherins, and are involved in cellto cell adhesions. A desmosome includes an extracellular core domain(the desmoglea) where the desmoglein and the desmocollin proteins ofadjacent cells bind one another. The adjacent cells are separated by alayer of ECM about 30 nanometers wide. Beneath the membrane, heavyplate-shaped structures are formed known as the outer dense plaque andthe inner dense plaque. The plaques are spanned by the desmoplakinprotein. The outer dense plaque is where the cytoplasmic domains of thecadherins attach to desmoplakin via plakoglobin and plakophillin. Theinner dense plaque is where desmoplakin attaches to the intermediatefilaments of the cell.

Interaction between individual cells and structures of thenanotopography may induce the passage of an agent through a barrier celland encourage transcellular transport. For instance, interaction withkeratinocytes of the stratum corneum can encourage the partitioning ofan agent into the keratinocytes, followed by diffusion through the cellsand across the lipid bilayer again. While an agent may cross a barrieraccording to both paracellular and transcellular routes, thetranscellular route may be predominate for highly hydrophilic molecules,though, of course, the predominant transport path may vary dependingupon the nature of the agent, hydrophilicity being only one definingcharacteristic.

According to one embodiment, the increased permeability of a cellularlayer can be determined through determination of the transepithelialelectrical resistance (TEER, also referred to herein as transepithelialresistance or TER) across the layer. As in generally known, a decreasein TEER across a cellular layer is a good indication of an increase inthe permeability of a cell layer due to, for instance, the lack offormation or the opening of tight junctions of a cell layer. It is clearthat in endothelia and certain epithelia, the ability of tight junctionsto restrict paracellular flux is not immutable. Rather, this gatefunction of tight junctions is capable of dynamic regulation (Madara,1988; Rubin, 1992). In particular, the activation of signal transductionpathways either by receptor ligands or specific membrane-permeantmodulators can have striking effects on the permeability of theparacellular pathway. For example, protein kinase C activation causes asubstantial increase in the permeability of tight junctions in MDCKcells (Ojakian, 1981), an epithelial cell line. Cyclic AMP elevationdecreases permeability in brain endothelial cells in culture, a modelsystem for the study of the blood-brain barrier (Rubin et al., 1991).Cyclic AMP also decreases tight junction permeability in peripheralendothelial cells (Stelzner et al., 1989; Langeier et al., 1991).

The permeability properties of the tight junction also depend upon theintegrity of the adherens junction. Disruption of the adherens junctionby removal of extracellular Ca²⁺ leads to an opening of tight junctionsin MDCK cells (see Martinez-Palomo et al., 1980; Gumbiner and Simons,1986) and in endothelial cells (Rutten et al., 1987). Protein kinasesappear to be involved in this indirect modulation of tight junctionalintegrity in MDCK cells (Citi, 1992). The Ca²⁺-sensitive components ofthe adherens junction complex are the cadherins (reviewed by Geiger andAvalon, 1992), These transmembrane proteins mediate intercellularadhesiveness in a Ca²⁺-dependent, homophilic manner via theirextracellular domains. The cytoplasmic domain of the cadherinsassociates with three further proteins termed α-, β- and γ-catenin(Ozawa et al., 1989), which link the cadherins to the actin cytoskeletonand are required for cadherin adhesiveness (Hirano et al., 1987;Nagaruchi and Takeichi, 1988; Ozawa et al., 1990; Kintner, 1992; seeStappert and Kemler, 1993).

According to the present disclosure, contact between an epithelial layerand a device surface that includes a predetermined pattern of structuresfabricated on the surface, at least a portion of which are fabricated ona nanometer scale, can increase the permeability and decrease the TEERof the epithelial layer. For instance, the TEER of an epithelial layercan drop to less than about 95%, less than about 85%, or less than about70% of its initial value following contact between the layer and ananopatterned surface for a period of time. By way of example, followingabout 30 minutes of contact between an epithelial layer and a surfaceincluding a pattern of nanostructures thereon, the TEER across the layercan be between about 90% and about 95% of its initial value. Following60 minutes, the TEER across the layer can be between about 80% and about90% of its initial value, and following 120 minutes, the TEER across thelayer can be between about 60% and about 75% of its initial value.

FIG. 10 illustrates one method for determining the TEER across anepithelial layer. In this embodiment, a cell layer, for instance anepithelial cell monolayer, can be grown or otherwise located at the baseof an apical chamber. The base of the apical chamber can be amicroporous filter membrane, as shown, to allow flow between the twochambers and prevent individual cells from passing into the basalchamber. By way of example, the microporous filter membrane can includepores of about 0.4 micrometers at a density of about 4×10⁶pores/centimeter. Of course, the specific parameters of the varioussystem components are not critical, and can be varied as is known in theart. The apical chamber can be defined by a transwell insert that canfit into a larger well, as shown, thereby defining the basal chamber ofthe system. During use a first electrode 1 and a second electrode 2 canbe located on either side of the epithelial cell monolayer and anohmmeter 4 can be connected between the two electrodes. The ohmmeter 4can provide the TEER across the cell monolayer. Systems for determiningthe TEER across a cell layer are known, for instance, the Millicell™electrical resistance system (available from Millipore of Bedford,Mass.) can be utilized.

Contact between an epithelial cell layer and a surface includingnanostructures fabricated thereon can lead to a drop in TEER, whichindicates an increase in layer permeability. Accordingly, followingcontact between an epithelial layer and a nanostructured surface, theability to transport compounds across the layer can be greatlyincreased. This can improve transdermal delivery of compounds thatpreviously could not be delivered efficiently in this fashion. Forexample, the transdermal delivery of high molecular weight compounds,lipophilic compounds and/or charged compounds can be improved throughutilization of disclosed methods and devices.

Of course, nano-sized structures of the device may directly orindirectly affect other components of a surrounding microenvironment,and provided herein are only a representative sample of such structures.

The device including a fabricated nanotopography on a surface of thedevice may be formed according to a single-step process. Alternatively,a multi-step process may be used, in which a pattern of nanostructuresare fabricated on a pre-formed surface. For example, an array ofmicroneedles may be first formed and then a random or non-random patternof nanostructures may be fabricated on the surface of the formedmicroneedles. In either the single-step or two-step process, structuresmay be fabricated on a surface or on a mold surface according to anysuitable nanotopography fabrication method including, withoutlimitation, nanoimprinting, injection molding, lithography, embossingmolding, and so forth.

In general, an array of microneedles may be formed according to anystandard microfabrication technique including, without limitation,lithography; etching techniques, such as wet chemical, dry, andphotoresist removal; thermal oxidation of silicon; electroplating andelectroless plating; diffusion processes, such as boron, phosphorus,arsenic, and antimony diffusion; ion implantation; film deposition, suchas evaporation (filament, electron beam, flash, and shadowing and stepcoverage), sputtering, chemical vapor deposition (CVD), epitaxy (vaporphase, liquid phase, and molecular beam), electroplating, screenprinting, lamination, stereolithography, laser machining, and laserablation (including projection ablation).

An electrochemical etching process may be utilized in whichelectrochemical etching of solid silicon to porous silicon is used tocreate extremely fine (on the order of 0.01 μm) silicon networks thatmay be used as piercing structures. This method may use electrolyticanodization of silicon in aqueous hydrofluoric acid, potentially incombination with light, to etch channels into the silicon. By varyingthe doping concentration of the silicon wafer to be etched, theelectrolytic potential during etching, the incident light intensity, andthe electrolyte concentration, control over the ultimate pore structuremay be achieved. The material not etched (i.e. the silicon remaining)forms the microneedles.

Plasma etching may also be utilized, in which deep plasma etching ofsilicon is carried out to create microneedles with diameters on theorder of 0.1 micrometer or larger. Needles may be fabricated indirectlyby controlling the voltage (as in electrochemical etching).

Lithography techniques, including photolithography, e-beam lithography,X-ray lithography, and so forth may be utilized for primary patterndefinition and formation of a master die. Replication may then becarried out to form the device including an array of microneedles.Common replication methods include, without limitation, solvent-assistedmicromolding and casting, embossing molding, injection molding, and soforth. Self-assembly technologies including phase-separated blockcopolymer, polymer demixing and colloidal lithography techniques mayalso be utilized in forming a nanotopography on a surface.

Combinations of methods may be used, as is known. For instance,substrates patterned with colloids may be exposed to reactive ionetching (RIE, also known as dry etching) so as to refine thecharacteristics of a fabricated nanostructure such as nanopillardiameter, profile, height, pitch, and so forth. Wet etching may also beemployed to produce alternative profiles for fabricated nanostructuresinitially formed according to a different process, e.g., polymerde-mixing techniques.

Structure diameter, shape, and pitch may be controlled via selection ofappropriate materials and methods. For example, etching of metalsinitially evaporated onto colloidal-patterned substrates followed bycolloidal lift-off generally results in prism-shaped pillars. An etchingprocess may then be utilized to complete the structures as desired.Ordered non-spherical polymeric nanostructures may also be fabricatedvia temperature-controlled sintering techniques, which form a variety ofordered trigonal nanometric features in colloidal interstices followingselective dissolution of polymeric nanoparticles. These and othersuitable formation processes are generally known in the art (see, e.g.,Wood, J R Soc interface, 2007 Feb. 22; 4(12): 1-17, incorporated hereinby reference).

Other methods as may be utilized in forming a microneedle including afabricated nanotopography on a surface include nanoimprint lithographymethods utilizing ultra-high precision laser machining techniques,examples of which have been described by Hunt, et al. (U.S. Pat. No.6,995,336) and Guo, et al. (U.S. Pat. No. 7,374,864), both of which areincorporated herein by reference. Nanoimprint lithography is anano-scale lithography technique in which a hybrid mold is utilizedwhich acts as both a nanoimprint lithography mold and a photolithographymask. A schematic of a nanoimprint lithography technique is illustratedin FIGS. 11A-11C. During fabrication, a hybrid mold 30 imprints into asubstrate 32 via applied pressure to form features (e.g., microneedlesdefining nanotopography) on a resist layer (FIG. 11A). In general, thesurface of the substrate 32 may be heated prior to engagement with themold 30 to a temperature above its glass transition temperature (T_(g)).While the hybrid mold 30 is engaged with the substrate 32, a flow ofviscous polymer may be forced into the mold cavities to form features 34(FIG. 11B). The mold and substrate may then be exposed to ultravioletlight. The hybrid mold is generally transmissive to UV radiation savefor certain obstructed areas. Thus, the UV radiation passes throughtransmissive portions and into the resist layer. Pressure is maintainedduring cooling of the mold and substrate. The hybrid mold 30 is thenremoved from the cooled substrate 32 at a temperature below T₉ of thesubstrate and polymer (FIG. 11C).

To facilitate the release of the nanoimprinted substrate 32 includingfabricated features 34 from the mold 30, as depicted in FIG. 11C, it isadvantageous to treat the mold 30 with a low energy coating to reducethe adhesion with the substrate 32, as a lower surface energy of themold 30 and the resulting greater surface energy difference between themold 30, substrate 32, and polymer may ease the release between thematerials. By way of example, a silicon mold coating may be used such astrideca-(1,1,2,2-tetrahydro)-octytrichloro silane (F₁₃-TCS).

A nanoimprinting process is a dynamic one which includes filling a moldfollowed by detachment of a formed polymer from the mold. To fill themold features, the polymer temperature must be raised to a level highenough to initiate flow under the applied pressure. The higher thetemperature, the lower the polymer viscosity, and the faster and easierthe mold will fill. A higher pressure will also improve the fill rateand overall fill for better mold replication. To release thenanoimprinted substrate from the mold, the substrate temperature may belowered to a point where the yield strength exceeds the adhesionalforces exerted by the mold. By varying the temperature it is alsopossible to draw the polymer features during detachment to obtaindifferent structures, for instance structures as illustrated in FIG. 8.

Structures may also be formed according to chemical addition processes.For instance, film deposition, sputtering, chemical vapor deposition(CVD), epitaxy (vapor phase, liquid phase, and molecular beam),electroplating, and so forth can be utilized for building structures ona surface.

Self-assembled monolayer processes as are known in the art can beutilized to form a pattern of structures on a surface. For instance, theability of block copolymers to self-organize can be used to form amonolayer pattern on a surface. The pattern can then be used as atemplate for the growth of desired structures, e.g., colloids, accordingto the pattern of the monolayer.

By way of example, a two-dimensional, cross-linked polymer network canbe produced from monomers with two or more reactive sites. Suchcross-linked monolayers have been made using self-assembling monolayer(SAM) (e.g., a gold/alkyl thiol system) or Langmuir-Blodgett (LB)monolayer techniques (Ahmed et al., Thin Solid Films 187: 141-153(1990)) as are known in the art. The monolayer can be crosslinked, whichcan lead to formation of a more structurally robust monolayer.

The monomers used to form a patterned monolayer can incorporate all thestructural moieties necessary to affect the desired polymerizationtechnique and/or monolayer formation technique, as well as to influencesuch properties as overall solubility, dissociation methods, andlithographic methods. A monomer can contain at least one, and more oftenat least two, reactive functional groups.

A molecule used to form an organic monolayer can include any of variousorganic functional groups interspersed with chains of methylene groups.For instance a molecule can be a long chain carbon structure containingmethylene chains to facilitate packing. The packing between methylenegroups can allow weak Van der Waals bonding to occur, enhancing thestability of the monolayer produced and counteracting the entropicpenalties associated with forming an ordered phase. In addition,different terminal moieties, such as hydrogen-bonding moieties may bepresent at one terminus of the molecules, in order to allow growth ofstructures on the formed monolayer, in which case the polymerizablechemical moieties can be placed in the middle of the chain or at theopposite terminus. Any suitable molecular recognition chemistry can beused in forming the assembly. For instance, structures can be assembledon a monolayer based on electrostatic interaction, Van der Waalsinteraction, metal chelation, coordination bonding (i.e., Lewisacid/base interactions), ionic bonding, covalent bonding, or hydrogenbonding.

When utilizing a SAM-based system, an additional molecule can beutilized to form the template. This additional molecule can haveappropriate functionality at one of its termini in order to form a SAM.For example, on a gold surface, a terminal thiol can be included. Thereare a wide variety of organic molecules that may be employed to effectreplication. Topochemically polymerizable moieties, such as dienes anddiacetylenes, are particularly desirable as the polymerizing components.These can be interspersed with variable lengths of methylene linkers.

For an LB monolayer, only one monomer molecule is needed because themolecular recognition moiety can also serve as the polar functionalgroup for LB formation purposes. Lithography can be carried out on a LBmonolayer transferred to a substrate, or directly in the trough. Forexample, an LB monolayer of diacetylene monomers can be patterned by UVexposure through a mask or by electron beam patterning.

Monolayer formation can be facilitated by utilizing molecules thatundergo a topochemical polymerization in the monolayer phase. Byexposing the assembling film to a polymerization catalyst, the film canbe grown in situ, and changed from a dynamic molecular assembly to amore robust polymerized assembly.

Any of the techniques known in the art for monolayer patterning may beused for patterning of the monolayer. Techniques useful in patterning amonolayer include, but are not limited to, photolithography, e-beamtechniques, focused ion-beam techniques, and soft lithography. Variousprotection schemes such as photoresist can be used for a SAM-basedsystem. Likewise, block copolymer patterns can be formed on gold andselectively etched to form patterns. For a two-component system,patterning can also be achieved with readily available techniques.

Soft lithography techniques can be utilized to pattern the monolayer inwhich ultraviolet light and a mask can be used for patterning. Forinstance, an unpatterned base monolayer may be used as a platform forassembly of a UV/particle beam reactive monomer monolayer. The monomermonolayer may then be patterned by UV photolithography, e-beamlithography, or ion beam lithography, even though the base SAM is notpatterned.

Growth of structures on a patterned monolayer can be achieved by variousgrowth mechanisms, such as through appropriate reduction chemistry of ametal salts and the use of seed or template-mediated nucleation. Usingthe recognition elements on the monolayer, inorganic growth can becatalyzed at this interface by a variety of methods. For instanceinorganic compounds in the form of colloids bearing the shape of thepatterned organic monolayer can be formed. For instance calciumcarbonate or silica structures can be templated by various carbonylfunctionalities such as carboxylic acids and amides. By controlling thecrystal growth conditions, it is possible to control the thickness andcrystal morphology of the mineral growth. Titanium dioxide can also betemplated.

Templated electroless plating techniques can be used to synthesizemetals using existing organic functional groups. In particular, bychelating metal atoms to the carbonyl moieties of the organic pattern,electroless metal deposition can be catalyzed on the pattern, formingpatterned metallic colloids. For instance, Cu, Au, Ni, Ag, Pd, Pt andmany other metals plateable by electroless plating conditions may beused to form metal structures in the shape of the organic monolayer. Bycontrolling the electroless plating conditions, it is possible tocontrol the thickness of the plated metal structures.

Other ‘bottom-up’ type growth methods as are known in the art can beutilized, for example a method as described in U.S. Pat. No. 7,189,435Tuominen, et al., which is incorporated herein by reference, can beutilized. According to this method, a conducting or semiconductingsubstrate (for example, a metal, such as gold) can be coated with ablock copolymer film (for example, a block copolymer ofmethylmethacrylate and styrene), where one component of the copolymerforms nanoscopic cylinders in a matrix of another component of thecopolymer. A conducting layer can then be placed on top of the copolymerto form a composite structure. Upon vertically orientation of thecomposite structure, some of the first component can be removed, forinstance by exposure to UV radiation, an electron beam, or ozone,degradation, or the like to form nanoscopic pores in that region of thesecond component.

In another embodiment, described in U.S. Pat. No. 6,926,953 to Nealey,et al., incorporated herein by reference, copolymer structures can beformed by exposing a substrate with an imaging layer thereon, forinstance an alkylsiloxane or an octadecyltrichlorosilane self assembledmonolayer, to two or more beams of selected wavelengths to forminterference patterns at the imaging layer to change the wettability ofthe imaging layer in accordance with the interference patterns. A layerof a selected block copolymer, for instance a copolymer of polystyreneand poly(methyl methacrylate) can then be deposited onto the exposedimaging layer and annealed to separate the components of the copolymerin accordance with the pattern of wettability and to replicate thepattern of the imaging layer in the copolymer layer. Stripes or isolatedregions of the separated components may thus be formed with periodicdimensions in the range of 100 nanometers or less.

The device surface can include a random distribution of fabricatednanostructures. Optionally, the surface can include additionalmaterials, in conjunction with the fabricated nanostructures. Forexample, a microneedle surface may have fabricated thereon anelectrospun fibrous layer, and a random or non-random pattern ofstructures may be fabricated on this electrospun layer.

Electrospinning includes of the use of a high voltage supplier to applyan electrical field to a polymer melt or solution held in a capillarytube, inducing a charge on the individual polymer molecules. Uponapplication of the electric field, a charge and/or dipolar orientationwill be induced at the air-surface interface. The induction causes aforce that opposes the surface tension. At critical field strength, theelectrostatic forces will overcome surface tension forces, and a jet ofpolymer material will be ejected from the capillary tube toward aconductive, grounded surface. The jet is elongated and accelerated bythe external electric field as it leaves the capillary tube. As the jettravels in air, some of the solvent may evaporate, leaving behindcharged polymer fibers which may be collected on the surface. As thefibers are collected, the individual and still wet fibers may adhere toone another, forming a nonwoven web on the surface. A pattern ofnanostructures may then be fabricated on the electrospun surface, forinstance through an embossing technique utilizing a mold defining thedesired nanostructures. Applying the mold to the microneedle surface atsuitable temperature and pressure can transfer the pattern to themicroneedle surface. A surface of random electrospun nano-sized fibersmay further improve the desirable characteristics of a microneedlesurface, e.g., one or more of surface area to volume ratio, surfaceroughness, surface energy, and so forth, and may provide associatedbenefits.

The surface of a microneedle surface can be further functionalized forimproved interaction with tissues or individual cells during use. Forinstance, one or more biomolecules such as polynucleotides,polypeptides, entire proteins, polysaccharides, and the like can bebound to a structured surface prior to use.

In some embodiments, a surface including structures formed thereon canalready contain suitable reactivity such that additional desiredfunctionality may spontaneously attach to the surface with nopretreatment of the surface necessary. However, in other embodiments,pretreatment of the structured surface prior to attachment of thedesired compound may be carried out. For instance, reactivity of astructure surface may be increased through addition or creation ofamine, carboxylic acid, hydroxy, aldehyde, thiol, or ester groups on thesurface. In one representative embodiment, a microneedle surfaceincluding a pattern of nanostructures formed thereon may be aminatedthrough contact with an amine-containing compound such as3-aminopropyltriethoxy silane in order to increase the aminefunctionality of the surface and bind one or more biomolecules to thesurface via the added amine functionality.

Materials as may be desirably bound to the surface of a patterned devicecan include ECM proteins such as laminins, tropoelastin or elastin,Tropocollagen or collagen, fibronectin, and the like. Short polypeptidefragments can be bound to the surface of a patterned device such as anRGD sequence, which is part of the recognition sequence of integrinbinding to many ECM proteins. Thus, functionalization of a microneedlesurface with RGD can encourage interaction of the device with ECMproteins and further limit foreign body response to the device duringuse.

Devices may be associated with an agent for delivery via the device. Forinstance, a transdermal microneedle patch may be utilized for deliveryof materials beneath the stratum corneum to the stratum spinosum or thestratum germinativum, or even deeper into the dermis. In general, anagent may be transported across the stratum corneum in conjunction withthe microneedle, e.g., within the microneedle or at the surface of themicroneedle.

The device may include a reservoir, e.g., a vessel, a porous matrix,etc., that may store and agent and provide the agent for delivery. Thedevice may include a reservoir within the device itself. For instance,the device may include a hollow, or multiple pores that may carry one ormore agents for delivery. The agent may be released from the device viadegradation of a portion or the entire device or via diffusion of theagent from the device.

FIGS. 12A and 12B are perspective views of the device including areservoir. The device 110 includes a reservoir 112 defined by animpermeable backing layer 114 and a microneedle array 116. The backinglayer and the microneedle array 116 are joined together about the outerperiphery of the device, as indicated at 118. The impermeable backinglayer 114 may be joined by an adhesive, a heat seal or the like. Thedevice 110 also includes a plurality of microneedles 120. A releaseliner 122 can be removed prior to use of the device to exposemicroneedles 120.

A formulation including one or more agents may be retained within thereservoir 112. Materials suitable for use as impermeable backing layer114 can include materials such as polyesters, polyethylene,polypropylene and other synthetic polymers. The material is generallyheat or otherwise sealable to the backing layer to provide a barrier totransverse flow of reservoir contents.

Reservoir 112, defined by the space or gap between the impermeablebacking layer 14 and the microneedle array 16, provides a storagestructure in which to retain the suspension of agents to beadministered. The reservoir may be formed from a variety of materialsthat are compatible with an agent to be contained therein. By way ofexample, natural and synthetic polymers, metals, ceramics, semiconductormaterials, and composites thereof may form the reservoir.

In one embodiment, the reservoir may be attached to the substrate uponwhich the microneedles are located. According to another embodiment, thereservoir may be separate and removably connectable to the microneedlearray or in fluid communication with the microneedle array, for instancevia appropriate tubing, luer locks, etc.

The device may include one or a plurality of reservoirs for storingagents to be delivered. For instance, the device may include a singlereservoir that stores a single or multiple agent-containing formulation,or the device may include multiple reservoirs, each of which stores oneor more agents for delivery to all or a portion of the array ofmicroneedles. Multiple reservoirs may each store a different materialthat may be combined for delivery. For instance, a first reservoir maycontain an agent, e.g., a drug, and a second reservoir may contain avehicle, e.g., saline. The different agents may be mixed prior todelivery. Mixing may be triggered by any means, including, for example,mechanical disruption (i.e. puncturing, degradation, or breaking),changing the porosity, or electrochemical degradation of the walls ormembranes separating the chambers. Multiple reservoirs may containdifferent active agents for delivery that may be delivered inconjunction with one another or sequentially.

In one embodiment, the reservoir may be in fluid communication with oneor more microneedles of the transdermal device, and the microneedles maydefine a structure (e.g., a central or lateral bore) to allow transportof delivered agents beneath the barrier layer.

In alternative embodiments, a device may include a microneedle assemblyand a reservoir assembly with flow prevention between the two prior touse. For instance, a device may include a release member positionedadjacent to both a reservoir and a microneedle array. The release membermay be separated from the device prior to use such that during use thereservoir and the microneedle array are in fluid communication with oneanother. Separation may be accomplished through the partial or completedetachment of the release member, For example, referring to FIGS. 13-18,one embodiment of a release member is shown that is configured to bedetached from a transdermal patch to initiate the flow of a drugcompound. More particularly, FIGS. 13-14 show a transdermal patch 300that contains a drug delivery assembly 370 and a microneedle assembly380. The drug delivery assembly 370 includes a reservoir 306 positionedadjacent to a rate control membrane 308.

The rate control membrane may help slow down the flow rate of the drugcompound upon its release. Specifically, fluidic drug compounds passingfrom the drug reservoir to the microneedle assembly via microfluidicchannels may experience a drop in pressure that results in a reductionin flow rate. If this difference is too great, some backpressure may becreated that may impede the flow of the compound and potentiallyovercome the capillary pressure of the fluid through the microfluidicchannels, Thus, the use of the rate control membrane may ameliorate thisdifference in pressure and allow the drug compound to be introduced intothe microneedle at a more controlled flow rate. The particularmaterials, thickness, etc. of the rate control membrane may vary basedon multiple factors, such as the viscosity of the drug compound, thedesired delivery time, etc.

The rate control membrane may be fabricated from permeable,semi-permeable or microporous materials that are known in the art tocontrol the rate of drug compounds and having permeability to thepermeation enhancer lower than that of drug reservoir. For example, thematerial used to form the rate control membrane may have an average poresize of from about 50 nanometers to about 5 micrometers, in someembodiments from about 100 nanometers to about 2 micrometers, and insome embodiments, from about 300 nanometers to about 1 micrometer (e.g.,about 600 nanometers). Suitable membrane materials include, forinstance, fibrous webs (e.g., woven or nonwoven), apertured films,foams, sponges, etc., which are formed from polymers such aspolyethylene, polypropylene, polyvinyl acetate, ethylene n-butyl acetateand ethylene vinyl acetate copolymers. Such membrane materials are alsodescribed in more detail in U.S. Pat. Nos. 3,797,494, 4,031,894,4,201,211, 4,379,454, 4,436,741, 4,588,580, 4,615,699, 4,661,105,4,681,584, 4,698,062, 4,725,272, 4,832,953, 4,908,027, 5,004,610,5,310,559, 5,342,623, 5,344,656, 5,364,630, and 6,375,978, which areincorporated in their entirety herein by reference for all relevantpurposes. A particularly suitable membrane material is available fromLohmann Therapie-Systeme.

Referring to FIGS. 13-14, although optional, the assembly 370 alsocontains an adhesive layer 304 that is positioned adjacent to thereservoir 306. The microneedle assembly 380 likewise includes a support312 from which extends a plurality of microneedles 330 having channels331, such as described above. The layers of the drug delivery assembly370 and/or the microneedle assembly 380 may be attached together ifdesired using any known bonding technique, such as through adhesivebonding, thermal bonding, ultrasonic bonding, etc.

Regardless of the particular configuration employed, the patch 300 alsocontains a release member 310 that is positioned between the drugdelivery assembly 370 and the microneedle assembly 380. While therelease member 310 may optionally be bonded to the adjacent support 312and/or rate control membrane 308, it is typically desired that it isonly lightly bonded, if at all, so that the release member 310 may beeasily withdrawn from the patch 300. If desired, the release member 310may also contain a tab portion 371 (FIGS. 13-14) that extends at leastpartly beyond the perimeter of the patch 300 to facilitate the abilityof a user to grab onto the member and pull it in the desired direction.In its “inactive” configuration as shown in FIGS. 13-14, the drugdelivery assembly 370 of the patch 300 securely retains a drug compound307 so that it does not flow to any significant extent into themicroneedles 330. The patch may be “activated” by simply applying aforce to the release member so that it is detached from the patch.

Referring to FIGS. 15-16, one embodiment for activating the patch 300 isshown in which the release member 310 is pulled in a longitudinaldirection. The entire release member 310 may be removed as shown inFIGS. 17-18, or it may simply be partially detached as shown in FIGS.15-16. In either case, however, the seal previously formed between therelease member 310 and the aperture (not shown) of the support 312 isbroken. In this manner, a drug compound 107 may begin to flow from thedrug delivery assembly 170 and into the channels 131 of the microneedles130 via the support 112. An exemplary illustration of how the drugcompound 307 flows from the reservoir 306 and into the channels 331 isshown in FIGS. 17-18. Notably, the flow of the drug compound 307 ispassively initiated and does not require any active displacementmechanisms (e.g., pumps).

In the embodiments shown in FIGS. 13-18, the detachment of the releasemember immediately initiates the flow of the drug compound to themicroneedles because the drug delivery assembly is already disposed influid communication with the microneedle assembly. In certainembodiments, however, it may be desired to provide the user with agreater degree of control over the timing of the release of the drugcompound. This may be accomplished by using a patch configuration inwhich the microneedle assembly is not initially in fluid communicationwith the drug delivery assembly. When it is desired to use the patch,the user may physically manipulate the two separate assemblies intofluid communication. The release member may be separated either beforeor after such physical manipulation occurs.

Referring to FIGS. 19-24, for example, one particular embodiment of apatch 200 is shown. FIGS. 19-20 illustrate the patch 200 before use, andshows a first section 250 formed by a microneedle assembly 280 and asecond section 260 formed by a drug delivery assembly 270. The drugdelivery assembly 270 includes a reservoir 206 positioned adjacent to arate control membrane 208 as described above. Although optional, theassembly 270 also contains an adhesive layer 204 that is positionedadjacent to the reservoir 206. The microneedle assembly 280 likewiseincludes a support 212 from which extends a plurality of microneedles230 having channels 231, such as described above.

In this embodiment, the support 212 and the rate control membrane 208are initially positioned horizontally adjacent to each other, and arelease member 210 extends over the support 212 and the rate controlmember 208. In this particular embodiment, it is generally desired thatthe release member 210 is releasably attached to the support 212 and therate control membrane 208 with an adhesive (e.g., pressure-sensitiveadhesive). In its “inactive” configuration as shown in FIGS. 19-20, thedrug delivery assembly 270 of the patch 200 securely retains a drugcompound 207 so that it does not flow to any significant extent into themicroneedles 230. When it is desired to “activate” the patch, therelease member 210 may be peeled away and removed, such as illustratedin FIGS. 21-22, to break the seal previously formed between the releasemember 210 and the aperture (not shown) of the support 212. Thereafter,the second section 260 may be folded about a fold line “F” as shown bythe directional arrow in FIG. 23 so that the rate control member 208 ispositioned vertically adjacent to the support 212 and in fluidcommunication therewith. Alternatively, the first section 250 may befolded. Regardless, folding of the sections 250 and/or 260 initiates theflow of a drug compound 207 from the drug delivery assembly 270 and intothe channels 231 of the microneedles 230 via the support 212 (See FIG.24).

The device may deliver an agent at a rate so as to be therapeuticallyuseful. In accord with this goal, a transdermal device may include ahousing with microelectronics and other micro-machined structures tocontrol the rate of delivery either according to a preprogrammedschedule or through active interface with the patient, a healthcareprofessional, or a biosensor. The device may include a material at asurface having a predetermined degradation rate, so as to controlrelease of an agent contained within the device. A delivery rate may becontrolled by manipulating a variety of factors, including thecharacteristics of the formulation to be delivered (e.g., viscosity,electric charge, and/or chemical composition); the dimensions of eachdevice (e.g., outer diameter and the volume of any openings); the numberof microneedles on a transdermal patch; the number of individual devicesin a carrier matrix; the application of a driving force (e.g., aconcentration gradient, a voltage gradient, a pressure gradient); theuse of a valve; and so forth.

Transportation of agents through the device may be controlled ormonitored using, for example, various combinations of valves, pumps,sensors, actuators, and microprocessors. These components may beproduced using standard manufacturing or microfabrication techniques.Actuators that may be useful with the device may include micropumps,microvalves, and positioners. For instance, a microprocessor may beprogrammed to control a pump or valve, thereby controlling the rate ofdelivery.

Flow of an agent through the device may occur based on diffusion orcapillary action, or may be induced using conventional mechanical pumpsor nonmechanical driving forces, such as electroosmosis orelectrophoresis, or convection. For example, in electroosmosis,electrodes are positioned on a biological surface (e.g., the skinsurface), a microneedle, and/or a substrate adjacent a microneedle, tocreate a convective flow which carries oppositely charged ionic speciesand/or neutral molecules toward or into the delivery site.

Flow of an agent may be manipulated by selection of the material formingthe microneedle surface. For example, one or more large grooves adjacentthe microneedle surface of the device may be used to direct the passageof drug, particularly in a liquid state. Alternatively, the physicalsurface properties of the device may be manipulated to either promote orinhibit transport of material along the surface, such as by controllinghydrophilicity or hydrophobicity.

The flow of an agent may be regulated using valves or gates as is knownin the art. Valves may be repeatedly opened and closed, or they may besingle-use valves. For example, a breakable barrier or one-way gate maybe installed in the device between a reservoir and the patternedsurface. When ready to use, the barrier may be broken or gate opened topermit flow through to the microneedle surface. Other valves or gatesused in the device may be activated thermally, electrochemically,mechanically, or magnetically to selectively initiate, modulate, or stopthe flow of molecules through the device. In one embodiment, flow iscontrolled by using a rate-limiting membrane as a “valve.”

In general, any agent delivery control system, including reservoirs,flow control systems, sensing systems, and so forth as are known in theart may be incorporated with devices. By way of example, U.S. Pat. Nos.7,250,037, 7,315,758, 7,429,258, 7,582,069, and 7,611,481 describereservoir and control systems as may be incorporated in devices.

Agents as may be delivered by the device may be intended for the localarea near the device or may be intended for wider distribution. Forinstance, in one embodiment, the device may deliver agents for painmanagement or inflammation management to a local area around a joint,for instance in treatment of osteoarthritis or rheumatoid arthritis.

The nanotopography of the device may improve delivery of agents whileminimizing foreign body and immune response. This may prove particularlybeneficial when considering delivery of oligonucleotides and othertherapeutics to the nuclear envelope. In the past, delivery of materials(e.g., plasmids, siRNA, RNAi, and so forth), to the nuclear envelope hasproven problematic because even when endocytosis is achieved, properendosomal delivery to the nuclear envelope has proven difficult, mostlikely due to foreign body and immune response. Once in the cytoplasm,delivered material is often recycled via late endosomes or degraded inthe lysosome. According to disclosed methods, interaction of amicroneedle with the ECM may prevent foreign body response within a cellfollowing endocytosis and encourage delivery of the materials to thenucleus.

Delivery of protein therapeutics has likewise proven problematic. Forinstance, delivery of high molecular weight agents such as proteintherapeutics has proven difficult for transdermal delivery routes due tothe natural barriers of the skin. The presence of the nanotopography ona microneedle may beneficially affect the thermodynamics of the ECM andimprove efficiency of delivery and uptake of protein therapeutics. Asutilized herein, the term ‘protein therapeutics’ generally refers to anybiologically active proteinaceous compound including, withoutlimitation, natural, synthetic, and recombinant compounds, fusionproteins, chimeras, and so forth, as well as compounds including the 20standard amino acids and/or synthetic amino acids. For instance, thepresence of the device in or near the stratum granulosum can open tightjunctions and allow paracellular transport of high molecular weightagents. In one embodiment, the device may be utilized in transdermaldelivery of high molecular weight agents (e.g., agents defining amolecular weight greater than about 400 Da, greater than about 10 kDa,greater than about 20 kDa, or greater than about 100 kDa, e.g., about150 kDa). Additionally, variation of the surface area to volume ratio ofthe device may be utilized to alter protein adsorption at the surface ofthe device, which may in turn alter delivery and cellular uptake ofmaterials. Thus, deliver of a particular material may be optimizedthrough optimization of the surface area/volume ratio of the device.

Even when considering delivery of small molecular weight agents, thedevice may provide increased efficiency and improved uptake due tointeraction of the device with components of the dermal connectivetissue and accompanying decrease in foreign body response andimprovement in localized chemical potential of the area.

Of course, devices are not limited to targeted delivery of agents.Systemic deliver of agents is also encompassed herein as is withdrawalof an agent from a subject via the device.

There is no particular limitation to agents as may be delivered by useof the device. Agents may include proteinaceous agents such as insulin,immunoglobulins (e.g., IgG, IgM, IgA, IgE), TNF-α, antiviralmedications, and so forth; polynucleotide agents including plasmids,siRNA, RNAi, nucleoside anticancer drugs, vaccines, and so forth; andsmall molecule agents such as alkaloids, glycosides, phenols, and soforth, Agents may include anti-infection agents, hormones, drugs thatregulate cardiac action or blood flow, pain control, and so forth. Stillother substances which may be delivered in accordance with the presentdisclosure are agents useful in the prevention, diagnosis, alleviation,treatment, or cure of disease. A non-limiting listing of agents includesanti-Angiogenesis agents, anti-depressants, antidiabetic agents,antihistamines, anti-inflammatory agents, butorphanol, calcitonin andanalogs, COX-II inhibitors, dermatological agents, dopamine agonists andantagonists, enkephalins and other opioid peptides, epidermal growthfactors, erythropoietin and analogs, follicle stimulating hormone,glucagon, growth hormone and analogs (including growth hormone releasinghormone), growth hormone antagonists, heparin, hirudin and hirudinanalogs such as hirulog, IgE suppressors and other protein inhibitors,immunosuppressives, insulin, insulinotropin and analogs, interferons,interleukins, leutenizing hormone, leutenizing hormone releasing hormoneand analogs, monoclonal or polyclonal antibodies, motion sicknesspreparations, muscle relaxants, narcotic analgesics, nicotine,non-steroid anti-inflammatory agents, oligosaccharides, parathyroidhormone and analogs, parathyroid hormone antagonists, prostaglandinantagonists, prostaglandins, scopolamine, sedatives, serotonin agonistsand antagonists, sexual hypofunction, tissue plasminogen activators,tranquilizers, vaccines with or without carriers/adjuvants,vasodilators, major diagnostics such as tuberculin and otherhypersensitivity agents as described in U.S. Pat. No. 6,569,143 entitled“Method of Intradermally Injecting Substances”, the entire content ofwhich is incorporated herein by reference. Vaccine formulations mayinclude an antigen or antigenic composition capable of eliciting animmune response against a human pathogen or from other viral pathogens.

In one preferred embodiment, the device may be utilized in treatment ofa chronic condition, such as rheumatoid arthritis, to deliver a steadyflow of an agent, to a subject in need thereof. RA drugs that can bedelivered via disclosed devices can include symptom suppressioncompounds, such as analgesics and anti-inflammatory drugs including bothsteroidal and non-steroidal anti-inflammatory drugs (NSAID), as well asdisease-modifying antirheumatic drugs (DMARDs).

The device can include and deliver symptom suppression compounds, suchas analgesics and anti-inflammatory drugs, as well as DMARD compounds,including biological DMARDs. While not wishing to be bound to anyparticular theory, it is understood that the nanometer-scare structuresfabricated on the surface of the device improve deliver of the compoundsacross the dermal barrier. Through utilization of the device, RA drugscan be delivered at a steady concentration over a sustained period. Thedevice can prevent the initial burst of concentration common whenutilizing previously known methods for delivery of RA drugs, includingoral delivery and injection.

RA drugs as may be incorporated in the device can include, withoutlimitation, one or more analgesics, anti-inflammatories, DMARDs,herbal-based drugs, and combinations thereof. Specific compounds can, ofcourse, fall under one or more of the general categories describedherein. For instance, many compounds function as both an analgesic andan anti-inflammatory; herbal-based drugs can likewise function as aDMARD as well as an anti-inflammatory. Moreover, multiple compounds thatcan fall under a single category can be incorporated in the device. Forinstance, the device can include multiple analgesics, such asacetaminophen with codeine, acetaminophen with hydrocodone (vicodin),and so forth.

Examples of analgesics and/or NSAIDs as may be incorporated in thedevices include analgesics available over the counter (OTC) atrelatively low dosages including acetamide (acetaminophen orparacetamol), acetylsalicylic acid (aspirin), ibuprofen, ketoprofen,naproxen and naproxen sodium, and the like. Prescription analgesicsand/or anti-inflammatories as may be incorporated in the device caninclude, without limitation, OTC analgesics at concentrations requiringa prescription, celecoxib, sulindac, oxaprozin, salsalate, piroxicam,indomethacin, etodolac, meloxicam, nabumetone, keteroloc and ketorolactromethamine, tolmetin, diclofenac, diproqualone, and diflunisal.Narcotic analgesics can include codeine, hydrocodone, oxycodone,fentanyl, and propoxyphene.

The device can include one or more steroidal anti-inflammatorycompounds, primarily glucocorticoids, including, without limitation,cortisone, dexamethasone, prednisolone, prednisone, hydrocortisone,triamcinolone, and methylprednisolone, betamethasone, and aldosterone.

DMARDs as may be included in the device can encompass both smallmolecule drugs and biological agents. DMARDs may be chemicallysynthesized or may be produced through genetic engineering processes(e.g., recombinant techniques).

Chemically synthesized DMARDs encompassed herein include, withoutlimitation, azathioprine, cyclosporine (cyclosporin, cyclosporine A),D-penicillamine, gold salts (e.g., auranofin, Na-aurothiomalate(Myocrism), chloroquine, hydroxychloroquine, leflunomide, methotrexate,minocycline, sulphasalazine (sulfasalazine), and cyclophosphamide.Biological DMARDs include, without limitation, TNF-α blockers such asetanercept (Enbrel®), infliximab (Remicade®), adalimumab (Humira®),certolizamab pego (Cimzia®) and golumumab (Simponi™); IL-1 blockers suchas anakinra (Kineret®); monoclonal antibodies against B cells includingrituximab (Rituxan®); T cell costimulation blockers such as abatacept(Orencia®), and IL-6 blockers such as tocilizumab (RoActemra®,Actemra®); a calcineurin inhibitor such as tacrolimus (Prograf®).

The device can incorporate one or more herbal-based or othernaturally-derived drugs. For instance, Ayurvedic compounds such asboswellic acid (extract of Boswellia serrata) and curcumin (curcuminoidsfrom Curcuma longa), as well as other naturally derived compounds suchas glucosamine sulfate (produced by hydrolysis of crustaceanexoskeletons or fermentation of a grain) can be incorporated in thedevice.

The device can incorporate multiple RA drugs. For instance, the devicecan include a combination of DMARDs in addition to an analgesic and/oran anti-inflammatory drug. Common combinations of DMARDs include, forexample, methotrexate in combination with hydroxychloroquine,methotrexate in combination with sulfasalazine, sulfasalazine incombination with hydroxychloroquine, and all three of these DMARDstogether, i.e., hydroxychloroquine, methotrexate, and sulfasalazine.

The devices can beneficially incorporate large and/or small molecularweight compounds. For instance, in the past, transdermal delivery ofprotein therapeutics has proven problematic due to the natural barriersof the skin. While not wishing to be bound to any particular theory, thepresence of the nanotopography of a microneedle of the device maybeneficially interact with cells and ECM of the dermal barrier andimprove efficiency of delivery and uptake of protein therapeutics. Forinstance, the presence of the device in or near the stratum granulosumcan open tight junctions and allow paracellular transport of highmolecular weight agents. As utilized herein, the term high molecularweight agents generally refers to agents defining a molecular weightgreater than about 400 Da, greater than about 10 kDa, greater than about20 kDa, or greater than about 100 kDa).

Even when considering delivery of smaller molecular weight drugs, thedevice may provide increased efficiency and improved uptake due tointeraction of the device with components of the dermal connectivetissue and accompanying decrease in foreign body response andimprovement in localized chemical potential of the area. In addition,the device can deliver the drugs at a steady concentration over asustained period, which can be beneficial.

The present disclosure may be further understood with reference to theExamples provided below.

Example 1

Several different molds were prepared using photolithography techniquessimilar to those employed in the design and manufacture of electricalcircuits. Individual process steps are generally known in the art andhave been described

Initially, silicon substrates were prepared by cleaning with acetone,methanol, and isopropyl alcohol, and then coated with a 258 nanometer(nm) layer of silicon dioxide according to a chemical vapor depositionprocess.

A pattern was then formed on each substrate via an electron beamlithography patterning process as is known in the art using a JEOLJBX-9300FS EBL system. The processing conditions were as follows:

-   -   Beam current=11 nA    -   Acceleration voltage=100 kV    -   Shot pitch=14 nm    -   Dose=260 μC/cm²    -   Resist=ZEP520A, ˜330 nm thickness    -   Developer=n-amyl acetate    -   Development=2 min. immersion, followed by 30 sec. isopropyl        alcohol rinse.

A silicon dioxide etch was then carried out with an STS Advanced OxideEtch (AOE). Etch time was 50 seconds utilizing 55 standard cubiccentimeters per minute (sccm) He, 22 sccm CF₄, 20 sccm C₄F₈ at 4 mTorr,400 W coil, 200 W RIE and a DC Bias of 404-411 V.

Following, a silicon etch was carried out with an STS silicon oxide etch(SOE). Etch time was 2 minutes utilizing 20 sccm Cl₂ and 5 sccm Ar at 5mTorr, 600 W coil, 50 W RIE and a DC Bias of 96-102 V. The silicon etchdepth was 500 nanometers.

A buffered oxide etchant (BOE) was used for remaining oxide removal thatincluded a three minute BOE immersion followed by a deionized waterrinse.

An Obducat NIL-Eitre®6 nanoimprinter was used to form nanopatterns on avariety of polymer substrates. External water was used as coolant. TheUV module utilized a single pulsed lamp at a wave length of between 200and 1000 nanometers at 1.8 W/cm². A UV filter of 250-400 nanometers wasused. The exposure area was 6 inches with a maximum temperature of 200°C. and 80 Bar. The nanoimprinter included a semi-automatic separationunit and automatic controlled demolding.

To facilitate the release of the nanoimprinted films from the molds, themolds were treated with Trideca-(1,1,2,2-tetrahydro)-octytrichlorosilane(F₁₃-TCS). To treat a mold, the silicon mold was first cleaned with awash of acetone, methanol, and isopropyl alcohol and dried with anitrogen gas. A Petri dish was placed on a hot plate in a nitrogenatmosphere and 1-5 ml of the F₁₃-TCS was added to the Petri dish. Asilicon mold was placed in the Petri dish and covered for 10-15 minutesto allow the F₁₃-TCS vapor to wet out the silicon mold prior to removalof the mold.

Five different polymers as given in Table 2, below, were utilized toform various nanotopography designs.

TABLE 2 Glass Surface Transition Tensile Tension Temperature, Modulus(mN/m) Polymer T_(g) (K) (MPa) @20° C. Polyethylene 140-170 100-300 30Polypropylene 280 1,389 21 PMMA 322 3,100 41 Polystyrene 373 3,300 40Polycarbonate 423 2,340 43

Several different nanotopography patterns were formed, schematicrepresentations of which are illustrated in FIGS. 25A-25D. Thenanotopography pattern illustrated in FIG. 25E was a surface of a flatsubstrate purchased from NTT Advanced Technology of Tokyo, Japan. Thepatterns were designated DN1 (FIG. 25A), DN2 (FIG. 25B), DN3 (FIG. 25C),DN4 (FIG. 25D) and NTTAT2 (FIG. 25E). SEM images of the molds are shownin FIGS. 25A, 25B, and 25C, and images of the films are shown in FIGS.25D and 25E. FIG. 8 illustrates a nanopatterned film formed by use ofthe mold of FIG. 25A (DN1). In this particular film, the polymerfeatures were drawn by temperature variation as previously discussed.The surface roughness of the pattern of FIG. 25E was found to be 34nanometers.

The pattern illustrated in FIGS. 7C and 7D was also formed according tothis nanoimprinting process. This pattern included the pillars 72 andpillars 62, as illustrated. Larger pillars 72 were formed with a 3.5micrometer (μm) diameter and 30 μm heights with center-to-center spacingof 6.8 μm. Pillars 62 were 500 nanometers in height and 200 nanometersin diameter and a center-to-center spacing of 250 nanometers.

The nanoimprinting process conditions used with polypropylene films areprovided below in Table 3.

TABLE 3 Time (s) Temperature(C.) Pressure (Bar) 10 50 10 10 75 20 10 10030 420 160 40 180 100 40 180 50 40 180 25 40

Example 2

Films were formed as described above in Example 1 including variousdifferent patterns and formed of either polystyrene (PS) orpolypropylene (PP). The underlying substrate varied in thickness.Patterns utilized were DN2, DN3, or DN4 utilizing formation processes asdescribed in Example 1. The pattern molds were varied with regard tohole depth and feature spacing to form a variety of differently-sizedfeatures having the designated patterns. Sample no. 8 (designated BB1)was formed by use of a 0.6 μm millipore polycarbonate filter as a mold.A 25 μm polypropylene film was laid over the top of the filter and wasthen heated to melt such that the polypropylene could flow into thepores of the filter. The mold was then cooled and the polycarbonate molddissolved by use of a methylene chloride solvent.

SEMs of the formed films are shown in FIGS. 26-34 and thecharacteristics of the formed films are summarized in Table 4, below.

TABLE 4 Film Cross Surface Water Sample thickness Pattern SectionalFeature Aspect Roughness Fractal Contact No. FIG. Pattern Material (μm)Feature¹ Dimension² height³ Ratio (nm) Dimension Angle 1 26 DN3 PS 75 A1100 nm  520 nm 0.47 150 2.0 100° B 400 nm 560 nm 1.4 C 200 nm 680 nm3.4 2 27A, DN2 PP 5.0 n/a 200 nm 100 nm 0.5 16 2.15  91° 27B 3 28 DN2 PS75 n/a 200 nm  1.0 μm 5 64 2.2 110° 4 29 DN2 PP 25.4 n/a 200 nm 300 nm1.5 38 1.94 118° 5 30 DN3 PS 75 A 1100 nm  570 nm 0.52 21.1 1.98 100° B400 nm 635 nm 1.6 C 200 nm — — 6 31 DN4 PS 75 n/a 200 nm — — 30.6 2.04 80° 7 32 DN4 PP 25.4 n/a 200 nm — — 21.4 2.07 112° 8 33 BB1 PP 25.4 n/a600 nm  18 μm 30 820 2.17 110° 9 34 DN3 PP 5 A 1100 nm  165 nm 0.15 502.13 — B 400 nm  80 nm 0.2 C 200 nm  34 nm 0.17 ¹Pattern Features asshown on the FIGS. ²Cross sectional dimension values were derived fromthe mold and equated as an approximation of the maximum dimension of thestructures, although it should be understood that the actual dimensionof any given individual structure may vary slightly as may be seen inthe FIGS. ³Feature heights are provided as the average of severalindividually determined feature heights.

For each sample AFM was utilized to characterize the film.Characterizations included formation of scanning electron micrograph(SEM), determination of surface roughness, determination of maximummeasured feature height, and determination of fractal dimension.

The atomic force microscopy (AFM) probe utilized was a series 16 siliconprobe and cantilever available from μMasch. The cantilever had aresonant frequency of 170 kHz, a spring constant of 40 N/m, a length of230±5 μm, a width of 40±3 μm, and a thickness of 7.0±0.5 μm. The probetip was an n-type phosphorous-doped silicon probe, with a typical probetip radius of 10 nanometers, a full tip cone angle of 40°, a total tipheight of 20-25 μm, and a bulk resistivity 0.01-0.05 ohm-cm.

The surface roughness value given in Table 4 is the arithmetical meanheight of the surface area roughness parameter as defined in the ISO25178 series.

The Fractal Dimension was calculated for the different angles byanalyzing the Fourier amplitude spectrum; for different angles theamplitude Fourier profile was extracted and the logarithm of thefrequency and amplitude coordinates calculated. The fractal dimension,D, for each direction is then calculated as

D=(6+s)/2,

where s is the (negative) slope of the log-log curves. The reportedfractal dimension is the average for all directions.

The fractal dimension can also be evaluated from 2D Fourier spectra byapplication of the Log Log function. If the surface is fractal the LogLog graph should be highly linear, with at negative slope (see, e.g.,Fractal Surfaces, John C. Russ, Springer-Verlag New York, LLC, July,2008).

Example 3

HaCaT human skin epithelial cells were grown in DMEM, 10% FBS, 1%penicillin/streptomycin at 37° C., 5% CO₂ for 24 hours at aconcentration of 25,000 cell/cm² in 6 well plates. Plates either hadpolypropylene nanopatterned films formed as described above in Example 1and designate DN1, DN2 (Sample 4 of Table 4), DN3 or untreated surfaceat the bottom of the well. Nanopatterned films were adhered in placewith cyanoacrylate.

Cells were detached from the surfaces with 1 mL of trypsin per well for10 minutes, quenched with 1 mL growth medium (same as above), thentransferred to a microfuge tube and pelleted at 1200 rpm for 7 minutes.

RNA was isolated from pelleted cells using the RNeasy miniprep kit fromQiagen using the manufacturer's protocol. Briefly, cells were lysed,mixed with ethanol and spun down in a column. Lysates were then washed 3times, treated with DNase and eluted in 40 μl volumes.

cDNA was created from the RNA isolated using the RT first strand kitfrom SA Biosciences. Briefly, RNA was treated with DNase again at 42° C.for 5 minutes. Random primers and reverse transcriptase enzyme was thenadded and incubated at 42° C. for 15 minutes, then incubated at 95° C.for 5 minutes to stop reaction.

qPCR was then performed on the cDNA samples using RT profiler custom PCRarray from SA Biosciences with primers for IL1-β, IL6, IL8, IL10, IL1R1,TNFα, TGFβ-1, PDGFA, GAPDH, HDGC, RTC and PPC. Briefly, cDNA was mixedwith SYBR green and water, and then added to a PCR plate pre-fixed withthe correct sense and antisense primer pair for the gene of interest.The plate was then run on an ABI StepOnePlus PCR machine heated to 95°C. for 10 minutes, then for 45 cycles of: 15 seconds at 95° C. and 1minute at 60° C.

Delta delta C_(T) analysis was performed using GAPDH as the internalcontrol. HDGC, RTC and PPC levels were used as additional internalcontrols for activity and genomic DNA contamination.

One-way ANOVA and Tukey's 2-point tests were then used to determinestatistical significance in the differences between surfaces.

Table 5, below, presents the protein expressions obtained as the foldchange in expression on nanoimprinted structures produced onpolypropylene films versus expression on an unstructured film.

TABLE 5 Mold IL1-β IL6 IL8 IL10 IL1R1 TNFα TGFβ1 PDGFA DN1 2.24 3.330.36 1.17 0.6 0.57 0.37 1.37 DN2 3.18 3.2 0.46 0.43 0.36 0.57 0.42 1.23DN3 3.36 2.7 0.47 5.83 1.6 0.37 0.35 0.64

Example 4

Methods as described in Example 3 were utilized to examine theexpression level for several different cytokines from HaCaT human skinepithelial cells when the cells were allowed to develop on a variety ofdifferent polypropylene (PP) or polystyrene (PS) films, formed andpatterned as described above. The expression level for each cytokine wascompared to that from the same cell type cultured on standard tissueculture polystyrene (TCPS) and induced with lipopolysaccharide (LPS).Results are shown in Table 6, below

Cells developed on a polypropylene film nanopatterned with a DN2pattern, as described above (Sample 4 of Table 4), were found toupregulate expression of IL-1β, IL-1ra, IL-10, and MIP-1β downregulateexpression of IL-4, IL-13, MIG, KC, IL-2, MIP-1, TNF-α, IL-12, IL-16,and IL-1α as compared to TCPS.

Several other films were examined for effect on cellular expression ofdifferent cytokines. Films were designated as follows:

1—DN2 pattern on a 75 μm polystyrene film (Sample 3 of Table 4)

2—DN3 pattern on a 75 μm polystyrene film (Sample 1 of Table 4)

3—DN4 pattern on a 75 μm polystyrene film (Sample 6 of Table 4)

4—unimprinted 75 μm polystyrene film

5—DN2 pattern on a 25.4 μm polypropylene film (Sample 4 of Table 4)

6—DN4 pattern on a 25.4 μm polypropylene film (Sample 7 of Table 4)

7—DN2 pattern on a 5 μm polypropylene film (Sample 2 of Table 4)

8—BB1 polypropylene film (Sample 8 of Table 4)

9—unimprinted 25.4 μm polypropylene film

10—unimprinted 5 μm polypropylene film

Results are illustrated in Table 6, below. Results are provided asfollows:

-   -   −− expression level was below the testing threshold    -   − expression level was lower than that for TCPS    -   = expression level was similar to that for TCPS    -   + expression level was above that for TCPS, but below that when        induced with LPS    -   ++ expression level was similar to that for induction with LPS    -   +++ expression level was above that for induction with LPS

TABLE 6 Film 1 2 3 4 5 6 7 8 9 10 IL-1α −− −− −− −− −− −− −− −− −− −−IL-1β ++ −− −− ++ −− −− −− −− −− −− IL-12 = = = = = = = = = = TNF-α =+ == = = = = =+ = = MCP-1 =+ = = = = = = =+ = = IL-2 = =— =+ = = = = = −− =KC −− −— = = = = = = — — M1P-1α −− −− −− +++ −− −− + −− +++ +++ MIP-1b++ + = = = + = −−− −−− = MIG = −− = + −−− −−− −− −−− −−− = GM-CSI −− −−−− −− −− −− −− −− −− −− IL-4 −− −− −− −− −− −− −− −− −− −− IL-13 −− −−−− ++ −− −− −− −− −− −− IL-10 — — = = = = = = = =

Example 5

HaCaT human skin epithelial cells were grown in DMEM, 10% FBS, 1%penicillin/streptomycin at 37° C., 5% CO₂ for 24 hours at aconcentration of 25,000 cell/cm² in 6 well plates. Plates either had apolypropylene film formed as described above in Example 1 withdesignation DN1, DN2 (Sample 4 of Table 4), DN3 or an untreated surfaceat the bottom of the well. Films were adhered in place withcyanoacrylate.

Media was collected from each well and analyzed for cytokine productionwith a Milliplex Map Kit from Millipore. Beads to detect IL-1β, IL-1ra,IL-6, IL-8, IL-10, PDGF-AA, PGGF-AB/BB and TNF-α were used. Readingswere done on a BioRad BioPlex machine. Briefly, media was placed intomicroplate wells with filters. Primary beads were added and incubated atroom temperature for 1 hour with shaking. The plates were then washedand incubated with detection antibodies for 30 minutes at roomtemperature with shaking. Strepavidin-phycoerythrin was then added andincubated at room temperature for an additional 30 minutes. Plates werethen washed, beads were resuspended in assay buffer and medianfluorescent intensity was analyzed on the BioPlex.

Example 6

The permeability effects of films patterned as described herein weredetermined on a monolayer of Caco-2 cells (human epithelial colorectaladenocarcinoma cells).

Films formed as described above in Example 1 and Example 2 were utilizedincluding polypropylene (PP) or polystyrene (PS) films formed withpatterns designated as DN2, DN3, and DN4. A fourth film, designated asBB1 (described in Example 2, above) was also used. The protocol was runwith multiple examples of each film type.

The general protocol followed for each film was as follows:

Materials

Cell culture inserts 0.4 μm pore size HDPET membrane (BD Falcon)

24 well plate (BD Falcon)

Caco-2 media

Nanostructured membranes as described above

IgG-FITC (Sigma Aldrich)

BSA-FITC (Sigma Aldrich)

Minimum Essential Medium no phenol red (Invitrogen)

TEER voltmeter

Warmed PBS

Black 96-well plate

Aluminum foil

Protocol

-   -   1. Seed Caco-2 cells on collagen coated well inserts 2 weeks        before permeability assay is to be performed. Collagen coated        plates are made by making a 1:1 volume of 100% ethanol to        collagen. Dry surfaces in sterile hood overnight until dry.    -   2. Make 0.1 mg/mL solution of FITC-conjugated molecule (BSA,        IgG, etc) of interest in phenol red free Alpha MEM media. Wrap        in aluminum foil to protect from light.    -   3. Check for confluency of Caco-2 cells by measuring the        resistance. Resistance should be above ˜600 Ohms for confluency.    -   4. Aspirate old media from cell culture inserts on apical and        basolateral sides. Rinse with PBS to remove any residual        phenol-red dye.    -   5. Add 0.5 mL of FITC-conjugated solution on apical side of each        insert.    -   6. In another 24 well plate with cell culture inserts, add 0.5        mL of warmed PBS.    -   7. Transfer inserts to the plate with PBS. Blot the bottom of        the insert on a Kim wipe to remove residual phenol red.    -   8. t=0-time point: sample 75 μL from the basolateral side of        insert and transfer to a black-bottom 96-well plate. Replace the        volume with 75 μL of warmed PBS. Record the resistance of each        well using the “chopstick” electrodes.    -   9. Carefully add the membrane to the appropriately labeled well.        Controls are the unimprinted membranes and the cells alone.        Check under a microscope that the membranes make direct contact        to the cells. You should be able to see a sharp circle,        indicating contact with the cells.    -   10. t=0 time point: repeat step 7 and then place in the        incubator for 1 hour    -   11. t=1 time point: repeat step 7 and then place in the        incubator for 1 hour    -   12. t=2 time point: repeat step 7    -   13. Measure fluorescence signal using a spectrofluorometer plate        reader. FITC (excitation=490 nanometers, emission=520        nanometers)

Results

Films utilized and results obtained are summarized in Table 7, below.

TABLE 7 Sample no. (see Table 4) 2 3 4 5 6 7 8 Pattern DN2 DN2 DN2 DN3DN4 DN4 BB1 Material PP PS PP PS PS PP PP Effective 5.3 16.3 0.29 10.432.3 4.8 7.8 Compression Modulus (MPa) Effective Shear 5.32 58.9 218 31977.8 4.4 26.7 Modulus (MPa) BET Surface — 0.11 — 0.44 — 4.15 — Area(m²/g) BSA — 2 1.9 3.3 2 1.4 1 permeability increase at 120 min. (MW 66kDa) IgG permeability — 1 — 1 3.5 — — increase at 120 min. (MW 150 kDa)

Moduli were determined according to standard methods as are known in theart as described by Schubert, et al. (Sliding induced adhesion of stiffpolymer microfiber arrays: 2. Microscale behaviour, Journal RoyalSociety, Interface, Jan. 22, 2008. 10.1098/rsif.2007.1309)

The contact angles were measured by placing a drop of water on thesurface according to standard practice. (See, e.g., Woodward, First TenAngstroms, Portsmouth, Va.).

FIG. 35 graphically illustrates the effects on permeability to bovineserum albumin (BSA) in a monolayer of cells on polystyrene filmspatterned with nanopatterns as described herein. The film patternsincluded a DN2 pattern (sample no. 3), a DN3 pattern (sample no. 5), anda DN4 pattern (sample no. 6), as indicated. Also shown are results for anon-patterned PS film (marked PSUI on FIG. 35) and a layer of cells withno adjacent film (marked ‘cells’ on FIG. 35). The results areillustrated as fold increase in permeability as a function of timemeasured in hours.

FIG. 36A and FIG. 36B graphically illustrate the effects on permeabilityto immunoglobulin-G (IgG) in a monolayer of cells on polystyrene filmspatterned with nanopatterns as described herein. The film patternsincluded a DN2 pattern (sample no. 3), a DN3 pattern (sample no. 5), anda DN4 pattern (sample no. 6), as indicated. Also shown are results for anon-patterned film (marked PSUI on FIGS. 36A and 36B) and a layer ofcells with no adjacent film (marked ‘cells’ on FIGS. 36A and 36B). Thetwo figures show the data over two different time scales.

The BSA signal was read on a fluorometer and the IgG signal was read ona spectrophotometer.

FIGS. 37A and 37B are 3D live/dead flourescein staining images showingparacellular and transcellular transport of IgG across a monolayer ofcells on a polystyrene DN4 patterned surface (sample no. 6).

FIG. 38 graphically illustrates the effects on permeability to BSA in amonolayer of cells on polypropylene films patterned with nanopatterns asdescribed herein. Patterns included BB1 (sample no. 8), DN2 (sample no.4), and DN4 (sample no. 7), as indicated. Also shown are results for anon-patterned film (marked PPUI on FIG. 38) and a layer of cells with noadjacent film (marked ‘cells’ on FIG. 38).

FIG. 39 graphically illustrates the effects on permeability to IgG in amonolayer of cells on polypropylene films patterned with nanopatterns asdescribed herein. Patterns included BB1 (sample no. 8), DN2 (sample no.4), and DN4 (sample no. 7), as indicated. Also shown are results for anon-patterned film (marked PSUI on FIG. 39) and a layer of cells with noadjacent film (marked ‘cells’ on FIG. 39).

FIGS. 40A and 40B are 3D live/dead flourescein staining images showingparacellular transport of IgG across a monolayer of cells on apolypropylene DN2 patterned surface (sample no. 4).

FIGS. 41A-41F are scanning electron microscopy (SEM) images of Caco-2cells cultured on nanopatterned surfaces. Specifically, FIGS. 41A and41B illustrate Caco-2 cells on a flat polystyrene control film. FIGS.41C and 41D illustrate Caco-2 cells on a polystyrene film patterned witha DN2 pattern (sample no. 3) as described above, and FIGS. 41E and 41Fillustrate Caco-2 cells on a polystyrene film patterned with a DN3pattern (sample no. 5) as described above.

Example 7

A method as described in Example 6 was utilized to examine thepermeability of a monolayer of Caco-2 cells cells to the fusion proteintherapeutic etanercept (marketed under the trade name as Enbrel®). FIG.42 graphically illustrates the results for cell layers grown on severaldifferent patterned substrates including both polypropylene (DN2PP—Sample 4 of Table 4) and polystyrene (DN2 PS—Sample 3 of Table 4 andDN3 PS—Sample 1 of Table 4) as well as an unimprinted polystyrenemembrane (PSUI) and a layer of cells with no membrane (cells). Resultsare shown as a fold change from initial permeability with time. FIG. 43illustrates the fold increase in permeability from initial t=0 at twohours (t=2) following addition of the membrane to the well for thesubstrates and cellular layer of FIG. 42.

Example 8

An array of microneedles including a nanopatterned surface was formed.Initially, an array of microneedles as illustrated in FIG. 2 was formedon a silicon wafer via a photolithography process. Each needle includedtwo oppositely placed side channels, aligned with one through-die holein the base of the needle (not visible on FIG. 2).

Microneedles were formed according to a typical micromachining processon a silicon based wafer. The wafers were layered with resist and/oroxide layers followed by selective etching (oxide etching, DRIE etching,iso etching), resist stripping, oxide stripping, and lithographytechniques (e.g., iso lithography, hole lithography, slit lithography)according to standard methods to form the array of microneedles.

Following formation of the microneedle array, a 5 μm polypropylene filmincluding a DN2 pattern formed thereon as described above in Example 1,the characteristics of which are described at sample 2 in Table 4, waslaid over the microneedle array. The wafer/film structure was held on aheated vacuum box (3 in. H₂O vacuum) at elevated temperature (130° C.)for a period of one hour to gently pull the film over the surface of themicroneedles while maintaining the nanopatterned surface of the film.

FIG. 44 illustrates the film over the top of the array of microneedles,and FIG. 45 is a closer view of a single needle of the array includingthe nanopatterned film overlaying the top of the needle.

Example 9

Transdermal patches including microneedle arrays formed as described inExample 8 were formed. Patches were formed with either a DN2 pattern ora DN3 pattern on the microneedle array. The films defining the patternsthat were applied to the microneedles are described in Table 8, below.Film 1 is equivalent to sample no. 2 of Table 4 and Film 2 is equivalentto sample no. 9 of Table 4.

TABLE 8 Property Film 1 Film 2 Pattern DN2 DN3 Material polypropylenepolypropylene Film Thickness  5 μm  5 μm Height of structures 100 nm 165nm, 80 nm, 34 nm Aspect ratio of 0.5 0.15, 0.2, 0.17 structures AverageSurface  16 nm 50 nm Roughness R_(A) Fractal Dimension 2.15 2.13

Control patches were also formed that had no pattern formed on the filmand subsequently applied to the array of microneedles. Transdermal andsubcutaneous formulations of etanercept (Enbrel®) were preparedaccording to instructions from the drug supplier. The subcutaneous doseformulation (for the positive control) was prepared to facilitate a 4mg/kg subcutaneous drug dose. The concentration of Enbrel® fortransdermal delivery was adjusted such that an intended dosing of 200mg/kg was achieved in a 24 hr period.

A total of 10 BALB/C mice (assigned designations #1-#10) were used inthe study, 8 were transdermally dosed with Enbrel® (group 1) and 2 weresubcutaneously dosed with Enbrel® (group 2) as described in Table 9,below. The transdermal patches were applied to shaved skin areas andholes formed near the microneedle tips upon application of the patch tothe skin.

TABLE 9 Blood Group Test Dose Dose Collection Animal No. Article DrugRoute Dose Level volume Time Points Number 1 Transdermal Enbrel ®Transdermal 5 mg/subject 0.2 ml Pre-patch #1, #5 patch 0.5 h  #2, #6  2h #3, #7  6 h #4, #8 24 h #2, #6 72 h #3, #7 2 subcutaneous Enbrel ®Subcutaneous 4 mg/kg 0.1 ml 24 h #9, #10 delivery

Transdermal patches used included both those defining a nanotopographyon the surface (DN2 and DN3 patterns, as described above), as well aspatches with no pattern of nanotopography.

Samples of whole blood were collected at the time points indicated inTable 8. Approximately 100 to 200 μl blood was taken via mandibularbleeding and then centrifuged at approximately 1300 rpm for 10 minutesin a refrigerated centrifuge (set at 4° C.). The resulting serum wasaspirated and transferred within 30 minutes of bloodcollection/centrifugation to appropriately labeled tubes. The tubes werefrozen and stored in the dark at ≦−70° C. until they were analyzed forlevels of Enbrel® using Human sTNF-receptor ELISA kit (R&D Systems cat#DRT200). The space time between two blood samplings on the same subjectwas 24 hours, to prevent unnecessary stress placed on the subject.

FIG. 46 graphically illustrates the average PK profile of thetransdermal patches that defined a nanotopography thereon. An average ofthe results for all patches including nanotopography were used torepresent the overall effect of incorporating a nanotopography inconjunction with a microneedle transdermal patch. As can be seen, theblood serum level rose rapidly to over 800 ng/mL/cm² of patch areawithin the first two hours of attachment. Following, the blood serumlevel gradually declined to negligible within 24 hours of attachment.The data used to develop FIG. 46 is provided below in Table 10.

TABLE 10 Blood serum Time (hr) concentration (ng/ml) 0 0 0.5 192.1 2249.25 6 24.4 24 7.2 65 4.0875

FIGS. 47 and 47 illustrate electron microscopy cross sectional views ofthe skin that was held in contact with the patches. The images weretaken after the patches were removed (72 hours post-attachment). Thesample of FIG. 47 was in contact with a patch including a nanotopographyon the surface. Specifically, a DN2 pattern, as described above, wasformed on the surface of the patch. The sample of FIG. 47B was held incontact with a transdermal patch that did not define a pattern ofnanotopography on the surface. As can be seen, the sample of FIG. 47Bshows signs of inflammation and a high density of macrophage presence.

Example 10

Transdermal patches including microneedle arrays formed as described inExample 8 were formed. Patches were formed with either a DN2 pattern ora DN3 pattern on the microneedle array as described in Table 8 ofExample 9. Control patches were also formed that had no pattern formedon the film subsequently applied to the array of microneedles.Transdermal and subcutaneous formulations of etanercept (Enbrel®) wereprepared according to instructions from the drug supplier.

Test subjects (rabbits) were transdermally dosed with Enbrel® or weresubcutaneously (SubQ) dosed with Enbrel®. Results are illustratedgraphically in FIG. 48, which provides the blood serum concentration inpg/ml as a function of time. The data used to develop FIG. 48 isprovided below in Table 11, below.

TABLE 11 No structure Time microneedle Subcutaneous DN2 Subcutaneous DN30 0.00 0.00 0.00 0.00 0.00 0.5 0.00 157.49 0.00 1611.21 0.00 2 0.003029.07 0.00 3504.92 497.17 6 0.00 3545.14 338.23 3699.24 796.64 12 0.003577.13 731.22 3571.80 1080.60 24 116.78 3778.71 785.49 3464.70 1924.2448 134.23 3416.73 638.18 3885.31 1006.95 72 88.68 3356.64 572.77 3803.421172.67

While the subject matter has been described in detail with respect tothe specific embodiments thereof, it will be appreciated that thoseskilled in the art, upon attaining an understanding of the foregoing,may readily conceive of alterations to, variations of, and equivalentsto these embodiments. Accordingly, the scope of the present disclosureshould be assessed as that of the appended claims and any equivalentsthereto.

1. A medical device comprising an array of microneedles that extendoutwardly from a support, wherein at least one of the microneedlescontains a plurality of nanostructures formed on a surface thereof, thenanostructures being arranged in a predetermined pattern.
 2. The medicaldevice of claim 1, wherein the nanostructures are in the form ofpillars.
 3. The medical device of claim 1, wherein at least a portion ofthe nanostructures have a cross-sectional dimension of less than about500 nanometers, and greater than about 5 nanometers.
 4. The medicaldevice of claim 1, wherein at least a portion of the nanostructures havea cross-sectional dimension of less than about 300 nanometers, andgreater than about 100 nanometers.
 5. The medical device of claim 1,wherein the nanostructures have approximately the same cross-sectionaldimension.
 6. The medical device of claim 1, wherein the pattern furtherincludes microstructures, wherein the nanostructures have across-sectional dimension smaller than the microstructures.
 7. Themedical device of claim 6, wherein the microstructures have across-sectional dimension of greater than about 500 nanometers and thenanostructures have a cross-sectional dimension of less than about 300nanometers.
 8. The medical device of claim 6, further comprising secondnanostructures having a cross-sectional dimension less than thecross-sectional dimension of the microstructures and greater than thecross-sectional dimension of the first nanostructures.
 9. The medicaldevice of claim 1, wherein at least a portion of the nanostructures havea center-to-center spacing of from about 50 nanometers to about 1micrometer.
 10. The medical device of claim 1, wherein the ratio of thecross sectional dimension of two adjacent nanostructures to thecenter-to-center spacing between those two structures is between about1:1 and about 1:4.
 11. The medical device of claim 1, wherein at least aportion of the nanostructures have an equidistant spacing.
 12. Themedical device of claim 1, where at least a portion of thenanostructures have a height of from about 10 nanometers to about 20micrometers.
 13. The medical device of claim 1, wherein at least aportion of the nanostructures have a height of from about 100 nanometersto about 700 nanometers.
 14. The medical device of claim 1, wherein atleast a portion of the nanostructures have an aspect ratio of from about0.15 to about
 30. 15. The medical device of claim 1, wherein at least aportion of the nanostructures have an aspect ratio of from about 0.2 toabout
 5. 16. The medical device of claim 1, wherein the pattern has afractal dimension of greater than about
 1. 17. The medical device ofclaim 1, wherein the pattern has a fractal dimension of from about 1.5to about 2.5.
 18. The medical device of claim 1, wherein the microneedlesurface containing the plurality of nanostructures has an averagesurface roughness between about 10 nanometers and about 200 nanometers.19. The medical device of claim 1, wherein the microneedle surfacecontaining the plurality of nanostructures has an effective compressionmodulus between about 4 MPa and about 320 MPa.
 20. The medical device ofclaim 1, wherein the microneedle surface containing the plurality ofnanostructures has an effective shear modulus between about 0.2 MPa andabout 50 MPa.
 21. The medical device of claim 1, wherein the microneedlesurface containing the plurality of nanostructures has a water contactangle between about 80° and about 150°.
 22. The medical device of claim1, further comprising a reservoir for holding a drug compound.
 23. Themedical device of claim 22, wherein the drug compound has a molecularweight of greater than about 100 kDa.
 24. The medical device of claim23, wherein the drug compound is a protein therapeutic.
 25. The medicaldevice of claim 22, wherein the drug compound is a TNF-α blocker. 26.The medical device of claim 22, wherein at least one of the microneedlescontains a channel for delivering the drug compound.
 27. A method fordelivering a drug compound to a subdermal location, the methodcomprising: penetrating the stratum corneum with a microneedle that isin fluid communication with the drug compound, the microneedlecontaining a plurality of nanostructures formed on a surface thereof andarranged in a pattern; and transporting the drug compound through themicroneedle and across the stratum corneum.
 28. The method of claim 27,further comprising contacting an extracellular matrix protein, a plasmamembrane protein, or a combination thereof with the nanostructures. 29.The method of claim 28, wherein the plasma membrane protein is a focaladhesion protein, an endocytosis mediating receptor, or a transmembraneprotein.
 30. The method of claim 27, wherein the nanostructures changethe membrane conductivity of the cell.
 31. The method of claim 27,wherein the nanostructures change the structure of an intercellularjunction.
 32. The method of claim 31, wherein the intercellular junctionis a tight junction.
 33. The method of claim 27, wherein the drugcompound is delivered to the cytoplasm of a cell.
 34. The method ofclaim 27, wherein the drug compound is delivered to the nuclear envelopeof a cell.
 35. A medical device comprising a plurality of nanostructuresthat are fabricated on a surface of the medical device, the plurality ofnanostructures defining a fabricated nanotopography.
 36. The medicaldevice of claim 35, wherein the surface is a surface of a microneedle.37. The medical device of claim 36, wherein the microneedle is acomponent of a microneedle assembly.
 38. The medical device of claim 35,wherein at least a portion of the nanostructures have a cross-sectionaldimension of less than about 500 nanometers and greater than about 5nanometers, a center-to-center spacing of from about 50 nanometers toabout 1 micrometer, and a height of from about 10 nanometers to about 1micrometer.
 39. A method for forming a medical device comprisingfabricating a pattern of nanostructures on a surface of a microneedle.40. The method of claim 39, wherein the pattern of nanostructures arefabricated according to a techniques selected from the group consistingof photolithography, e-beam lithography, X-ray lithography,self-assembly techniques, reactive ion etching, wet etching, filmdeposition, sputtering, chemical vapor deposition, epitaxy,electroplating, and combinations thereof.
 41. The method of claim 39,wherein the step of fabricating the pattern of nanostructures comprisesa nanoimprint lithography technique.